Recombinant polypeptides, compositions, and methods thereof

ABSTRACT

The present disclosure provides recombinant polypeptides, homodimeric and heterodimeric proteins comprising the recombinant polypeptides, nucleic acid molecules encoding the recombinant polypeptides, and vectors and host cells comprising the nucleic acid molecules. The present disclosure also provides compositions comprising the recombinant polypeptides and methods of making and using the recombinant polypeptides.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:3902.001PC01_SeqListing_ST25.txt, Size: 134,451 bytes; and Date ofCreation: Dec. 21, 2017) submitted in this application is incorporatedherein by reference in its entirety.

BACKGROUND

Bone is a highly rigid tissue that constitutes part of the vertebralskeleton with unique mechanical properties derived from its extensivematrix structure. Throughout the life of animals, bone tissue iscontinuously renewed.

Processes of bone formation and renewal are carried out by specializedcells. Osteogenesis (bone formation or growth of bone) is carried out by“osteoblasts” (bone-forming cells). Bone remodeling occurs through aninterplay between the activities of bone-resorbing cells called“osteoclasts” and the bone-forming osteoblasts. Since these processesare carried out by specialized living cells, chemical (for example,pharmaceutical and/or hormonal), physical, and physicochemicalalterations can affect the quality, quantity, and shaping of bonetissue.

A variety of growth factors (for example, PDGF) as well as cytokines areinvolved in bone formation processes. It is thus valuable to identifyphysiologically acceptable chemical agents (for example, hormones,pharmaceuticals, growth factors, and cytokines) that can induce theformation of bone at a predetermined site. However, for the chemicalagents to be successfully used as therapeutic tools, several hurdlesneed to be overcome. One hurdle includes developing recombinantpolypeptides that have osteoinductive activity. For example,osteoinductive activity in recombinant human platelet-derived growthfactor-BB has not been demonstrated. Another hurdle is osteoinductivevariability in chemical agents. For example, demineralized bone matrix(DBM) is a chemical agent that is an osteoinductive allograft derivedfrom processed bone. An increasing number of DBM-based products arecommercially available, but osteoinductive variability has been foundacross different products and among production lots for the sameproduct. Thus, there is a need for chemical agents, such as recombinantpolypeptides and associated compositions, that demonstrate consistentosteoinductive activity.

BRIEF SUMMARY

The present disclosure is directed to a recombinant polypeptidecomprising: a first domain selected from the group consisting of SEQ IDNO: 35 and SEQ ID NO: 39; a second domain selected from the groupconsisting of SEQ ID NO: 47 and SEQ ID NO: 49; and a third domainselected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 61;wherein the first domain is fused to either C-terminal or N-terminal ofthe second domain, the third domain is fused to the second domain, thefirst domain or both of the first and the second domains, and whereinthe recombinant polypeptide is capable of inducing alkaline phosphataseactivity.

In certain embodiments, the first domain is located C-terminal to thesecond domain, the third domain is located N-terminal to the seconddomain, or the first domain is located N-terminal to the third domain.

In certain embodiments, the second domain of the recombinant polypeptidecomprises an intramolecular disulfide bond between the twenty-thirdamino acid of the second domain and the twenty-seventh amino acid of thesecond domain.

In certain embodiments, the third domain of the recombinant polypeptidecomprises a first amino acid sequence of PKACCVPTE (SEQ ID NO: 356) anda second amino acid sequence of GCGCR (SEQ ID NO: 357), wherein thethird domain comprises two intramolecular disulfide bonds between thefirst and second amino acid sequences.

In certain embodiments, the recombinant polypeptide comprises a firstintramolecular disulfide bond between the fourth amino acid of the firstamino acid sequence and the second amino acid of the second amino acidsequence, and a second intramolecular disulfide bond between the fifthamino acid of the first amino acid sequence and the fourth amino acid ofthe second amino acid sequence.

In certain embodiments, the recombinant polypeptide comprises a firstintramolecular disulfide bond between the fifth amino acid of the firstamino acid sequence and the second amino acid of the second amino acidsequence, and a second intramolecular disulfide bond between the fourthamino acid of the first amino acid sequence and the fourth amino acid ofthe second amino acid sequence.

In certain embodiments, the recombinant polypeptide is selected from thegroup consisting of: SEQ ID NO: 260, SEQ ID NO: 268, SEQ ID NO: 276, SEQID NO: 284, SEQ ID NO: 292, SEQ ID NO: 300, SEQ ID NO: 308, SEQ ID NO:316, SEQ ID NO: 324, SEQ ID NO: 332, SEQ ID NO: 340, and SEQ ID NO: 348.

The present disclosure is directed to a homodimeric protein comprisingtwo identical recombinant polypeptides of any of the above recombinantpolypeptides, wherein the homodimeric protein comprises anintermolecular disulfide bond between the first domains of the tworecombinant polypeptides.

In certain embodiments, the homodimeric protein comprises anintermolecular disulfide bond between the fifteenth amino acid in thefirst domain of one recombinant polypeptide and the fifteenth amino acidin the first domain of the other recombinant polypeptide.

In certain embodiments, the second domain of one or both of therecombinant polypeptides of the homodimeric protein comprises anintramolecular disulfide bond.

In certain embodiments, the third domain of each recombinant polypeptideof the homodimeric protein comprises a first amino acid sequence ofPKACCVPTE (SEQ ID NO: 356) and a second amino acid sequence of GCGCR(SEQ ID NO: 357), and the homodimeric protein comprises twointermolecular disulfide bonds between the first amino acid sequence inthe third domain of one recombinant polypeptide and the second aminoacid sequence in the third domain of the other recombinant polypeptide.In certain embodiments, the homodimeric protein comprises a firstintermolecular disulfide bond between the fourth amino acid of the firstamino acid sequence of the one recombinant polypeptide and the secondamino acid of the second amino acid sequence of the other recombinantpolypeptide, and a second intermolecular disulfide bond between thefifth amino acid of the first amino acid sequence of the one recombinantpolypeptide and the fourth amino acid of the second amino acid sequenceof the other recombinant polypeptide. In certain embodiments, thehomodimeric protein comprises a first intermolecular disulfide bondbetween the fifth amino acid of the first amino acid sequence of the onerecombinant polypeptide and the second amino acid of the second aminoacid sequence of the other recombinant polypeptide, and a secondintermolecular disulfide bond between the fourth amino acid of the firstamino acid sequence of the one recombinant polypeptide and the fourthamino acid of the second amino acid sequence of the other recombinantpolypeptide.

In certain embodiments, the third domain of each recombinant polypeptideof the homodimeric protein comprises a first amino acid sequence ofPKACCVPTE (SEQ ID NO: 356) and a second amino acid sequence of GCGCR(SEQ ID NO: 357), and the homodimeric protein comprises twointramolecular disulfide bonds between the first amino acid sequence inthe third domain of one recombinant polypeptide and the second aminoacid sequence in the third domain of the same recombinant polypeptide.In certain embodiments, the homodimeric protein comprises a firstintramolecular disulfide bond between the fourth amino acid of the firstamino acid sequence of the one recombinant polypeptide and the secondamino acid of the second amino acid sequence of the same recombinantpolypeptide, and a second intramolecular disulfide bond between thefifth amino acid of the first amino acid sequence of the one recombinantpolypeptide and the fourth amino acid of the second amino acid sequenceof the same recombinant polypeptide. In certain embodiments, thehomodimeric protein comprises a first intramolecular disulfide bondbetween the fifth amino acid of the first amino acid sequence of the onerecombinant polypeptide and the second amino acid of the second aminoacid sequence of the same recombinant polypeptide, and a secondintramolecular disulfide bond between the fourth amino acid of the firstamino acid sequence of the one recombinant polypeptide and the fourthamino acid of the second amino acid sequence of the same recombinantpolypeptide.

The present disclosure is directed to a heterodimeric protein comprisingtwo different recombinant polypeptides of any of the above recombinantpolypeptides, wherein the heterodimeric protein comprises anintermolecular disulfide bond between the first domains of the twodifferent recombinant polypeptides.

In certain embodiments, the heterodimeric protein comprises anintermolecular disulfide bond between the fifteenth amino acid in thefirst domain of one recombinant polypeptide and the fifteenth amino acidin the first domain of the other recombinant polypeptide.

In certain embodiments, the second domain of one or both of the twodifferent recombinant polypeptides of the heterodimeric proteincomprises an intramolecular disulfide bond.

The present disclosure is directed to a recombinant polypeptidecomprising an amino acid sequence selected from the group consisting of:SEQ ID NO: 260, SEQ ID NO: 268, SEQ ID NO: 276, SEQ ID NO: 284, SEQ IDNO: 292, SEQ ID NO: 300, SEQ ID NO: 308, SEQ ID NO: 316, SEQ ID NO: 324,SEQ ID NO: 332, SEQ ID NO: 340, and SEQ ID NO: 348, wherein therecombinant polypeptide is capable of inducing alkaline phosphataseactivity.

The present disclosure is directed to a composition comprising any ofthe above recombinant polypeptides, any of the above homodimericproteins, or any of the above heterodimeric proteins.

The present disclosure related to a sustained release compositioncomprising a calcium phosphate carrier, selected from the groupconsisting of tricalcium phosphate (TCP), alpha-tricalcium phosphate(α-TCP), beta-tricalcium phosphate (β-TCP), biphasic calcium phosphate(BCP) and mixture thereof; and a biodegradable matrix, selected from thegroup consisting of polylactic acid (PLA), polyglycolic acid (PGA),polylactic-co-glycolic acid (PLGA), Polyvinyl alcohol (PVA) and mixturethereof; and a homodimeric protein as previous mentioned. Furthermore,the sustained release composition comprises (a) about 2-11% (w/w) of thecalcium phosphate carrier; (b) about 88-97% (w/w) of the biodegradablematrix; and (c) about 0.017-0.039% (w/w) of the homodimeric protein.

The present disclosure related to a method of promoting healing of along-bone fracture in a subject in need of such treatment comprising:preparing a composition including a homodimeric protein as previousdescription homogeneously entrained within a slow release biodegradablecalcium phosphate carrier that hardens so as to impermeable to efflux ofthe homodimeric protein in vivo sufficiently that the long-bone fracturehealing is confined to the volume of the calcium phosphate carrier; andimplanting the composition at a location where the long-bone fractureoccurs, wherein the homodimeric protein is in an amount of from about0.03 mg/g to about 3.2 mg/g of the calcium phosphate carrier.

In certain embodiments, the method of promoting healing of a long-bonefracture further comprises gradually exposing the entrained homodimericprotein at the location as the calcium phosphate carrier degrades,wherein the calcium phosphate carrier has a calcium to phosphate ratioof about 0.4 to about 1.8.

The present disclosure related a biodegradable composition capable ofinducing bone growth to form a bone mass in a location comprises ahomodimeric protein as previous description; and a biodegradable calciumphosphate carrier having a plurality of pores. Further, the homodimericprotein is about 0.003-0.32% (w/w).

In certain embodiments, wherein a porosity of the biodegradable calciumphosphate carrier of the biodegradable composition is larger than 70%with pore size from about 300 μm to about 600 μm.

In certain embodiments, the biodegradable calcium phosphate carrier withpores that extend throughout the biodegradable calcium phosphatecarrier, wherein the homodimeric protein is in an effective amount offrom about 0.03 mg/g to about 3.2 mg/g of the biodegradable calciumphosphate carrier.

In certain embodiments, the biodegradable composition is suitable foraugmentation of a tissue selected from nasal furrows, frown lines,midfacial tissue, jaw-line, chin, and cheeks.

In certain embodiments, the location is selected from a long-bonefracture defect, a space between two adjacent vertebra bodies, anon-union bone defect, maxilla osteotomy incision, mandible osteotomyincision, sagittal split osteotomy incision, genioplasty osteotomyincision, rapid palatal expansion osteotomy incision, and a spaceextending lengthwise between two adjacent transverse processes of twoadjacent vertebrae.

In certain embodiments, the single dose of the homodimeric protein isfrom about 0.006 mg to about 15 mg.

In certain embodiments, the biodegradable calcium phosphate carrierhardens so as to be impermeable to efflux of the homodimeric protein invivo sufficiently that the formed bone mass is confined to the volume ofthe biodegradable calcium phosphate carrier.

The present disclosure related to a method for promoting arthrodesiscomprising administering the homodimeric protein as previous descriptionand a biodegradable calcium phosphate carrier to a malformed ordegenerated joint.

In certain embodiments, the step of administering the homodimericprotein includes administering from about 0.006 mg to about 10.5 mg ofthe homodimeric protein to the malformed or degenerated joint pertreatment.

The present disclosure related to a method for promoting spinal fusion,the method comprising the steps of exposing an upper vertebra and alower vertebra; identifying a site for fusion between the upper and thelower vertebra; exposing a bone surface on each of the upper and thelower vertebra at the site for fusion; and administering the homodimericprotein as previous description and a biodegradable calcium phosphatecarrier to the site.

In certain embodiments, the biodegradable calcium phosphate carrier is anon-compressible delivery vehicle, and wherein the non-compressibledelivery vehicle is for application to the site between the two bonesurfaces where bone growth is desired but does not naturally occur.

In certain embodiments, the biodegradable calcium phosphate carriercomprises at least one implantation stick for application to the sitesuch that the implantation stick extends lengthwise between the upperand the lower vertebrae.

The present disclosure related a spinal fusion device including abiodegradable composition as previous description; and a spinal fusioncage, configured to retain the biodegradable calcium phosphate carrier.

The present disclosure related to a method of generating a bone mass tofuse two adjacent vertebrae bodies in a spine of a subject in needincluding the steps of preparing a composition for generating the bonemass, and introducing the composition in a location between the twoadjacent vertebrae bodies. Furthermore, the composition including ahomodimeric protein as previous description that homogeneously entrainedwithin a slow release biodegradable carrier that hardens so as to beimpermeable to efflux of the homodimeric protein in vivo sufficientlythat the formed bone mass is confined to the volume of the slow releasebiodegradable carrier. The slow release biodegradable carrier graduallyexposes the entrained homodimeric protein at the location as the slowrelease biodegradable carrier degrades, and further wherein thehomodimeric protein is in an amount of from about 0.2 mg/site to about10.5 mg/site of the location.

In certain embodiments, the slow release biodegradable carrier has aporous structure, and cells from the two adjacent vertebrae migratesinto the porous structure so as to generate the bone mass.

In certain embodiments, the slow release biodegradable carrier has aninitial volume, and the bone mass replaces the initial volume of theslow release biodegradable carrier as the slow release biodegradablecarrier is resorbed.

The present disclosure related to a method for fusing adjacent vertebraebodies in a subject in need by a posterior or transforaminal fusionapproach including the steps of preparing a disc space for receipt of anintervertebral disc implant in an intervertebral space between theadjacent vertebrae; introducing a slow-release carrier including ahomodimeric protein as previous description into the intervertebral discimplant; and introducing the intervertebral disc implant in the discspace between the adjacent vertebrae for generating a bone mass in thedisc space. Furthermore, the homodimeric protein is in an amount of fromabout 0.2 mg/site to about 10.5 mg/site of the slow-release carrier

The present disclosure related to a moldable composition for filling anosseous void including a moldable matrix including about 90% to about99.5% by weight of the moldable composition; and the homodimeric proteinas previous description. Moreover, less than about 25% by percentage ofthe homodimeric protein is released from the moldable composition afterabout 1, 24, 48, 72, 168, 240 or about 336 hours post implantation.

The present disclosure related to a sustained release compositionincluding a calcium phosphate carrier; a biodegradable matrix; and arecombinant protein as previous description. Furthermore, the calciumphosphate carrier is about 2-11% (w/w), the biodegradable matrix isabout 88-97% (w/w), and the recombinant protein is about 0.017-0.039%(w/w).

In certain embodiments, the calcium phosphate carrier is selected fromthe group consisting of tricalcium phosphate (TCP), alpha-tricalciumphosphate (α-TCP), beta-tricalcium phosphate (β-TCP), biphasic calciumphosphate (BCP) and any combination thereof. In certain embodiments, thebiodegradable matrix is selected from the group consisting of polylacticacid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA),Polyvinyl alcohol (PVA) and any combination thereof.

The present disclosure related to a method for promoting healing of along-bone fracture in a subject including (a) preparing a compositionincluding a recombinant protein as previous description and abiodegradable calcium phosphate carrier; (b) hardening the composition;and (c) implanting the composition at a location. Furthermore, thelocation is an injury site in said subject where the long-bone fractureoccurs, and the protein is about 0.003-0.32% (w/w).

In certain embodiments, the method further includes (d) exposing thecomposition at the location as the calcium phosphate carrier degrades.In certain embodiments, the calcium phosphate carrier includes acalcium-to-phosphate ratio of about 0.4-1.8.

The present disclosure related to a method for promoting arthrodesis ina subject including the steps of administering a composition including arecombinant protein as previous description and a biodegradable calciumphosphate carrier to a location in said subject. Furthermore, an amountof the protein is about 0.006-10.5 mg, and the location is selected fromthe group consisting of a malformed joint, a degenerated joint andcombination thereof.

The present disclosure related to a method for promoting spinal fusionin a subject including (a) exposing an upper vertebra and a lowervertebra of said subject; (b) identifying a site for fusion between theupper and the lower vertebra; (c) exposing a bone surface on each of theupper and the lower vertebra at the site for fusion; and (d)administering a recombinant protein as previous describe and abiodegradable calcium phosphate carrier to the site.

In certain embodiments, the biodegradable calcium phosphate carrier is anon-compressible delivery vehicle capable of being applied to the sitewhere bone growth is desired but does not naturally occur.

In certain embodiments, the biodegradable calcium phosphate carrier inthe method for promoting spinal fusion includes an implantation stickextending lengthwise between the upper and the lower vertebrae.

The present disclosure related to a spinal fusion device including abiodegradable composition as previous mentioned; and a spinal fusioncage, configured to retain the biodegradable calcium phosphate carrier.

The present disclosure related to a method for generating a bone mass tofuse two adjacent vertebrae bodies in a spine of a subject including (a)preparing a composition for generating the bone mass, the compositionincluding a recombinant protein according to claim 9 and a slow releasebiodegradable carrier; (b) hardening the composition; (c) introducingthe composition between two adjacent vertebrae bodies; and (d) releasingthe composition and exposing the protein. Furthermore, the amount of theprotein is about 0.2-10.5 mg/site.

In certain embodiments, the slow release biodegradable carrier has aporous structure capable of receiving cells for generating a bone mass.

In certain embodiments, the slow release biodegradable carrier has aninitial volume, and the bone mass replaces the initial volume of theslow release biodegradable carrier as the slow release biodegradablecarrier is resorbed.

The present disclosure related to a method for fusing adjacent vertebraebodies in a subject in need including (a) preparing a disc space forreceipt of an intervertebral disc implant between adjacent vertebrae;(b) introducing a slow-release carrier including a recombinant proteinaccording to claim 9 into the intervertebral disc implant; (c)introducing the intervertebral disc implant in the disc space; and (d)generating a bone mass in the disc space. Furthermore, the amount of theprotein is about 0.2-10.5 mg/site.

The present disclosure related to a moldable composition forimplantation into an osseous void including a moldable matrix, and arecombinant protein as previous description. Furthermore, the amount ofthe moldable matrix is about 90-99.5% (w/w); and less than 25% of theprotein is released from the composition after a predetermined periodpost implantation.

In certain embodiments, the predetermined period is about 1, 24, 48, 72,168, 240 or 336 hours.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1B show representative X-ray images of female rabbit (strainNZW) ulnae for experimental Groups A-G. The ulna in each experimentalgroup contained a surgically created 20 mm-sized circumferential defect(i.e., a defect site). For Groups A-F, implants were made into thedefect sites. The ulna in each of Groups A-E received an implant of 200mg β-TCP. The β-TCP in Groups A, B, C, and D served as a carrier for 2,6, 20, and 60 μg, respectively, of a homodimeric protein including tworecombinant polypeptides (i.e., SEQ ID NO:260, including intramoleculardisulfide bond C44-C48). The β-TCP in Group E did not carry anyrecombinant polypeptide. Group F received an autograft implant of iliacbone fragments. Group G did not receive any implant into the defectsite. X-ray images were taken for each of Groups A-G at: 0 weeks (i.e.,immediately after surgery, “0 W”) and at 2, 4, 6, and 8 weeks aftersurgery (i.e., “2 W,” “4 W,” “6 W,” and “8 W,” respectively). The siteof the implant (Groups A-F) or the defect site (Group G) in the ulna islocated immediately above the white asterisk in each image.

FIGS. 2A to 2B show representative computerized tomography (CT) scanningimages for experimental Groups A-G. Change in cross-sectional imagesover time at the center of the implantation site (Groups A-F) or defectsite (Group G) is shown 0 weeks (i.e., immediately after surgery, “0 W”)and at 4 and 8 weeks after surgery (i.e., “4 W” and “8 W,”respectively). Groups A-G are as described for FIG. 1.

FIG. 3 shows a graphical representation of the results of torsionalstrength tests for an intact ulna that was not surgically altered andfor experimental Groups A-G (i.e., “A”-“G,” respectively). The maximumtorque in Newton-meters (“N-m”) is shown. Groups A-G are as describedfor FIG. 1.

FIGS. 4-9 show representative X-ray images of sheep spines forexperimental Groups 1-6. Groups 1-3 received a single implant of 3.5 gof β-TCP carrying 10.5, 3.5, or 1.05 mg, respectively, of a homodimericprotein including two recombinant polypeptides (i.e., SEQ ID NO:260,including intramolecular disulfide bonds C44-C48, C80-C112, andC79-C114). Group 4 received a single implant of 3.5 g of β-TCP withoutany homodimeric protein. Group 5 received a single implant of boneautograft. Group 6 received a single implant of absorbable collagensponge with 3.15 mg of rhBMP-2. FIGS. 4-9 show, from left to right ineach figure, radiographs taken post-operatively, at 4 weeks, and at 12weeks (harvest) from Groups 1-6, respectively. Groups 1-6 are designatedin FIGS. 4-9 as “2179,” “2192,” “2187,” “2160,” “2162,” and “2166,”respectively.

FIGS. 10a to 10y show representative X-ray images of female dog (strainbeagles) ulnae for five experimental groups (i.e., “Control,” “Hp 35,”“Hp 140,” “Hp 560,” and “Hp 2240,” respectively) taken at 1, 2, 4, 8,and 12 weeks after surgery (i.e., “1 W,” “2 W,” “4 W,” “8 W,” and “12W,” respectively). The ulna in each experimental group contained asurgically created 25 mm-sized circumferential defect (i.e., a defectsite). For the five groups, implants were made into the defect sites.The implants for the Control, Hp 35, Hp 140, Hp 560, and Hp 2240experimental groups were 800 mg of β-TCP carrying 0, 35, 140, 560, or2240 μg, respectively, of a homodimeric protein including tworecombinant polypeptides (i.e., SEQ ID NO:260, including intramoleculardisulfide bond C44-C48, and intermolecular disulfide bonds C80-C112 andC79-C114).

FIG. 11 shows a graphical representation of an interbody cage. The leftimages in each row show a top view of the interbody cage, while theright images show a side view. Magnification: top row=×0.67, middlerow=×2, bottom row=×4. Cage dimensions: about 8 mm×24 mm×10 mm.

FIG. 12 shows a graphical representation of the range of motion indegrees for: a sheep spine receiving an implant of 150 mg of β-TCPmeasured at 0 weeks (i.e., “Time Zero”); sheep spines receiving implantsof 150 mg of β-TCP carrying 0, 0.1, 0.5, 1.0, 2.0, or 4.0 mg,respectively, of a homodimeric protein including two recombinantpolypeptides (i.e., SEQ ID NO:260, including intramolecular disulfidebond C44-C48, and intermolecular disulfide bonds C79-C112 and C80-C114)measured at 12 weeks; and a sheep spine receiving an implant of boneautograft measured at 12 weeks. Sheep were female, strain Ewe. Thefigure shows the mean and standard deviations for the range of motionfor each testing condition and direction.

FIGS. 13A-C show representative micro computed tomography (μCT) axial,coronal, and sagittal images, respectively, of a sheep spine thatreceived an implant with homodimeric protein in an amount of 0.1 mg/siteas described for FIG. 12. “V,” “D,” “R,” “L,” “S,” and “I” refer toventral, dorsal, right, left, superior, and inferior directions,respectively. The site generated bone that largely filled the spacewithin the cage; however, lucency was present at both endplateinterfaces.

FIGS. 14A-C show representative μCT axial, coronal, and sagittal images,respectively, of a sheep spine for that received an implant withhomodimeric protein in an amount of 0.5 mg/site as described for FIG.12. “V,” “D,” “R,” “L,” “S,” and “I” are as described for FIGS. 13A-C.The site demonstrated good bone quality but with the presence of somelucent lines within the graft.

FIGS. 15A-C show representative μCT axial, coronal, and sagittal images,respectively, of a sheep spine that received an autograft as describedfor FIG. 12. “V,” “D,” “R,” “L,” “S,” and “I” are as described for FIGS.13A-C. The site did not fully fill the cage with bone at the time point.Additionally, there was some lucency within the endplate.

FIGS. 16a and 16b show the relative amount of the homodimeric proteinreleased (FIG. 16a ) and the cumulative percentage of the homodimericprotein released (FIG. 16b ) from microparticles over a 14-day releaseperiod.

FIG. 17 shows a representative scanning electron microscopy image ofpoly lacticco-glycolic acid/homodimeric protein-tricalcium phosphate(PLGA/Hp-β-TCP) stored at −20° C., 4° C. and 25° C. for six months.

FIG. 18 shows representative X-ray images of Balb/C mice tibias at 0weeks and after 4 weeks of implantation of PLGA/Hp-β-TCP as describedfor FIG. 17 and with different dosages of homodimeric protein. Whitearrows in each image identify the bone defects. (Groups=C: necrotic bonecontrol (i.e., bone fragment without implantation of any scaffold), PT:PLGA/β-TCP (i.e., no homodimeric protein), POT-0.2: PLGA/0.2 μgHp-β-TCP, POT-0.8: PLGA/0.8 Hp-β-TCP, POT-1.6: PLGA/1.6 μg Hp-β-TCP andPOT-3.2: PLGA/3.2 μg Hp-β-TCP).

FIG. 19 shows a graphical representation of the percentages of new boneformation/area in osteonecrosis bone after 4 weeks of implantation, withthe groups as described for FIG. 18.

FIGS. 20A-D show cross-section representations of example formulationsof the present disclosure: granules of a carrier (e.g., β-TCP) carryingpolypeptides/proteins of the disclosure (FIG. 20A), a putty mixed withthe granules carrying polypeptides/proteins of the disclosure (FIG.20B), a putty comprising polypeptides/proteins of the disclosure (FIG.20C), and a putty comprising polypeptides/proteins of the disclosurewith granules carrying polypeptides/proteins of the disclosuredistributed evenly in the outer layer of the putty (FIG. 20D).

FIG. 21 shows a graphical representation of the cumulative percentage ofhomodimeric protein released over a duration of specified hours.

DETAILED DESCRIPTION

Provided herein are recombinant polypeptides, homodimeric andheterodimeric proteins comprising the recombinant polypeptides, nucleicacid molecules and vectors encoding the recombinant polypeptides, andhost cells for expressing the recombinant polypeptides. The presentdisclosure also provides compositions of the recombinant polypeptidesand methods of making and using the recombinant polypeptides.

All publications cited herein are hereby incorporated by reference intheir entireties, including without limitation all journal articles,books, manuals, patent applications, and patents cited herein, to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. As used throughout the instantapplication, the following terms shall have the following meanings.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context dictatesotherwise. Thus, for example, reference to “a domain” includes a domainor a plurality of such domains and reference to “the recombinantpolypeptide” includes reference to one or more recombinant polypeptides,and so forth. The terms “a”, “an,” “the,” “one or more,” and “at leastone,” for example, can be used interchangeably herein.

The use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

“Amino acid” is a molecule having the structure wherein a central carbonatom (the alpha-carbon atom) is linked to a hydrogen atom, a carboxylicacid group (the carbon atom of which is referred to herein as a“carboxyl carbon atom”), an amino group (the nitrogen atom of which isreferred to herein as an “amino nitrogen atom”), and a side chain group,R. When incorporated into a peptide, polypeptide, or protein, an aminoacid loses one or more atoms of its amino acid carboxylic groups in thedehydration reaction that links one amino acid to another. As a result,when incorporated into a protein, an amino acid is referred to as an“amino acid residue.”

“Protein” or “polypeptide” refers to any polymer of two or moreindividual amino acids (whether or not naturally occurring) linked via apeptide bond, and occurs when the carboxyl carbon atom of the carboxylicacid group bonded to the alpha-carbon of one amino acid (or amino acidresidue) becomes covalently bound to the amino nitrogen atom of aminogroup bonded to the non alpha-carbon of an adjacent amino acid. The term“protein” is understood to include the terms “polypeptide” and “peptide”(which, at times may be used interchangeably herein) within its meaning.In addition, proteins comprising multiple polypeptide subunits (e.g.,DNA polymerase III, RNA polymerase II) or other components (for example,an RNA molecule, as occurs in telomerase) will also be understood to beincluded within the meaning of “protein” as used herein. Similarly,fragments of proteins and polypeptides are also within the scope of thedisclosure and may be referred to herein as “proteins.” In one aspect ofthe disclosure, a polypeptide comprises a chimera of two or moreparental peptide segments. The term “polypeptide” is also intended torefer to and encompass the products of post-translation modification(“PTM”) of the polypeptide, including without limitation disulfide bondformation, glycosylation, carbamylation, lipidation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, modification by non-naturally occurringamino acids, or any other manipulation or modification, such asconjugation with a labeling component. A polypeptide can be derived froma natural biological source or produced by recombinant technology, butis not necessarily translated from a designated nucleic acid sequence.It can be generated in any manner, including by chemical synthesis. An“isolated” polypeptide or a fragment, variant, or derivative thereofrefers to a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan simply be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for the purpose of the disclosure, as are nativeor recombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Domain” as used herein can be used interchangeably with the term“peptide segment” and refers to a portion or fragment of a largerpolypeptide or protein. A domain need not on its own have functionalactivity, although in some instances, a domain can have its ownbiological activity.

A particular amino acid sequence of a given protein (i.e., thepolypeptide's “primary structure” when written from the amino-terminusto the carboxyl-terminus) is determined by the nucleotide sequence ofthe coding portion of a mRNA, which is in turn specified by geneticinformation, typically genomic DNA (including organelle DNA, e.g.,mitochondrial or chloroplast DNA). Thus, determining the sequence of agene assists in predicting the primary sequence of a correspondingpolypeptide and more particular the role or activity of the polypeptideor proteins encoded by that gene or polynucleotide sequence.

“N-terminal” as used herein refers to position or location of an aminoacid, domain, or peptide segment within a polypeptide in relation to theamino-terminus of the polypeptide. For example, “domain A is N-terminalto domains B and C” means that domain A is located closer to theamino-terminus than domains B and C, such that the order of domains inthe polypeptide from the amino-terminus is understood to be either A-B-Cor A-C-B when the locations of domains B and C are not otherwisespecified. Additionally, any number of amino acids, including none, canbe present between a domain that is N-terminal to another domain.Similarly, any number of amino acids, including none, can be presentbetween the N-terminus of the polypeptide and a domain that isN-terminal the other domains in the polypeptide.

“C-terminal” as used herein refers to position or location of an aminoacid, domain, or peptide segment within a polypeptide in relation to thecarboxyl-terminus of the polypeptide. For example, “domain A isC-terminal to domains B and C” means that domain A is located closer tothe carboxyl-terminus than domains B and C, such that the order ofdomains in the polypeptide from the amino-terminus is understood to beeither B-C-A or C-B-A when the locations of domains B and C are nototherwise specified. Additionally, any number of amino acids, includingnone, can be present between a domain that is C-terminal to anotherdomain. Similarly, any number of amino acids, including none, can bepresent between the C-terminus of the polypeptide and a domain that isC-terminal the other domains in the polypeptide.

“Intramolecular” and “intermolecular” used herein when referring todisulfide bonds, refer to disulfide bonds that occur within apolypeptide chain and between polypeptide chains, respectively.

“Fused,” “operably linked,” and “operably associated” are usedinterchangeably herein when referring to two or more domains to broadlyrefer to any chemical or physical coupling of the two or more domains inthe formation of a recombinant polypeptide as disclosed herein. In oneembodiment, a recombinant polypeptide as disclosed herein is a chimericpolypeptide comprising a plurality of domains from two or more differentpolypeptides.

Recombinant polypeptides comprising two or more domains as disclosedherein can be encoded by a single coding sequence that comprisespolynucleotide sequences encoding each domain. Unless stated otherwise,the polynucleotide sequences encoding each domain are “in frame” suchthat translation of a single mRNA comprising the polynucleotidesequences results in a single polypeptide comprising each domain.Typically, the domains in a recombinant polypeptide as described hereinwill be fused directly to one another or will be separated by a peptidelinker. Various polynucleotide sequences encoding peptide linkers areknown in the art.

“Homodimeric protein”, “heterodimeric protein”, and “homodimeric orheterodimeric protein” as used herein refers to a protein having tworecombinant polypeptides that are identical or different. Therefore, the“homodimeric protein”, “heterodimeric protein”, and “homodimeric orheterodimeric protein” as used herein also refers to the “homodimericrecombinant protein”, “heterodimeric recombinant protein”, and“homodimeric or heterodimeric recombinant protein.” Furthermore,“recombinant protein” as used herein refers to “homodimeric protein”,“heterodimeric protein”, or “homodimeric or heterodimeric protein”.

“Polynucleotide” or “nucleic acid” as used herein refers to a polymericform of nucleotides. In some instances, a polynucleotide comprises asequence that is either not immediately contiguous with the codingsequences or is immediately contiguous (on the 5′ end or on the 3′ end)with the coding sequences in the naturally occurring genome of theorganism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides of the disclosurecan be ribonucleotides, deoxyribonucleotides, or modified forms ofeither nucleotide. A polynucleotide as used herein refers to, amongothers, single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. The term polynucleotide encompasses genomic DNA or RNA(depending upon the organism, i.e., RNA genome of viruses), as well asmRNA encoded by the genomic DNA, and cDNA. In certain embodiments, apolynucleotide comprises a conventional phosphodiester bond or anon-conventional bond (e.g., an amide bond, such as found in peptidenucleic acids (PNA)). By “isolated” nucleic acid or polynucleotide isintended a nucleic acid molecule, e.g., DNA or RNA, which has beenremoved from its native environment. For example, a nucleic acidmolecule comprising a polynucleotide encoding a recombinant polypeptidecontained in a vector is considered “isolated” for the purposes of thepresent disclosure. Further examples of an isolated polynucleotideinclude recombinant polynucleotides maintained in heterologous hostcells or purified (partially or substantially) from otherpolynucleotides in a solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present disclosure.Isolated polynucleotides or nucleic acids according to the presentdisclosure further include polynucleotides and nucleic acids (e.g.,nucleic acid molecules) produced synthetically.

As used herein, a “coding region” or “coding sequence” is a portion of apolynucleotide, which consists of codons translatable into amino acids.Although a “stop codon” (TAG, TGA, or TAA) is typically not translatedinto an amino acid, it may be considered to be part of a coding region,but any flanking sequences, for example promoters, ribosome bindingsites, transcriptional terminators, introns, and the like, are not partof a coding region. The boundaries of a coding region are typicallydetermined by a start codon at the 5′ terminus, encoding theamino-terminus of the resultant polypeptide, and a translation stopcodon at the 3′ terminus, encoding the carboxyl-terminus of theresulting polypeptide.

As used herein, the term “expression control region” refers to atranscription control element that is operably associated with a codingregion to direct or control expression of the product encoded by thecoding region, including, for example, promoters, enhancers, operators,repressors, ribosome binding sites, translation leader sequences,introns, polyadenylation recognition sequences, RNA processing sites,effector binding sites, stem-loop structures, and transcriptiontermination signals. For example, a coding region and a promoter are“operably associated” if induction of promoter function results in thetranscription of mRNA comprising a coding region that encodes theproduct, and if the nature of the linkage between the promoter and thecoding region does not interfere with the ability of the promoter todirect the expression of the product encoded by the coding region orinterfere with the ability of the DNA template to be transcribed.Expression control regions include nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding region, and which influence the transcription,RNA processing, stability, or translation of the associated codingregion. If a coding region is intended for expression in a eukaryoticcell, a polyadenylation signal and transcription termination sequencewill usually be located 3′ to the coding sequence.

“Nucleic acid segment,” “oligonucleotide segment” or “polynucleotidesegment” refers to a portion of a larger polynucleotide molecule. Thepolynucleotide segment need not correspond to an encoded functionaldomain of a protein; however, in some instances the segment will encodea functional domain of a protein. A polynucleotide segment can be about6 nucleotides or more in length (e.g., 6-20, 20-50, 50-100, 100-200,200-300, 300-400 or more nucleotides in length).

A “vector” refers to any vehicle for the cloning of and/or transfer of anucleic acid molecule into a host cell. The term “vector” includes bothviral and nonviral vehicles (e.g., plasmid, phage, cosmid, virus) forintroducing the nucleic acid into a cell in vitro, ex vivo or in vivo.

As used herein, the terms “host cell” and “cell” can be usedinterchangeably and can refer to any type of cell or a population ofcells, e.g., a primary cell, a cell in culture, or a cell from a cellline, that harbors or is capable of harboring a nucleic acid molecule(e.g., a recombinant nucleic acid molecule). Host cells can be aprokaryotic cell, or alternatively, the host cells can be eukaryotic,for example, fungal cells, such as yeast cells, and various animalcells, such as insect cells or mammalian cells.

“Culture,” “to culture” and “culturing,” as used herein, means toincubate cells under in vitro conditions that allow for cell growth ordivision or to maintain cells in a living state. “Cultured cells,” asused herein, means cells that are propagated in vitro.

“Osteoinductive” as used herein refers to the induction of bone and/orcartilage formation or growth, including, for example, the induction ofa marker associated with bone and/or cartilage formation or growth(e.g., the induction of alkaline phosphatase activity).

“Yeast two-hybrid assay” or “yeast two-hybrid system” is usedinterchangeably herein and refers to an assay or system for thedetection of interactions between protein pairs. In a typical two-hybridscreening assay/system, a transcription factor is split into twoseparate fragments, the binding domain (BD) and the activation domain(AD), each of which is provided on a separate plasmid, and each of whichis fused to a protein of interest. The yeast two-hybrid assay systemcomprises (i) a “bait” vector, comprising a bait protein and the BD ofthe transcription factor utilized in the system; (ii) a “prey” vector,comprising a prey protein (or a library of prey proteins to be screenedfor interaction with the bait protein) and the AD of the transcriptionfactor; and (iii) a suitable reporter yeast strain containing thebinding sequence for the BD of the transcription factor used in thesystem. When the bait-prey interaction occurs, the AD of thetranscription factor drives the expression of one or more reporterproteins. The bait and prey vectors are introduced into the reporteryeast strain, wherein the expressed bait and prey proteins may interact.Alternatively, separate haploid yeast strains each containing either abait vector or a prey vector can be mated and the resulting diploidyeast strain expresses both proteins. Interacting bait and prey proteinpairs result in the reconstitution and activation of the transcriptionfactor, which then binds to its compatible activation domain provided inthe reporter yeast strain, which in turn triggers the expression of thereporter gene, which may then be detected.

Recombinant Polypeptides and Compositions

The present disclosure is directed to a recombinant polypeptidecomprising any two or more domains selected from the group consisting ofSEQ ID NOs: 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, and 355, including, without limitation,any of the combinations of two domains disclosed in Table 3 herein. Incertain embodiments, the recombinant polypeptide comprises any three ofthe domains, including, without limitation to, any of the combinationsof three domains disclosed in Table 3, herein.

Any domain of a recombinant polypeptide as described herein can belocated at any position with respect to the amino-terminus orcarboxyl-terminus of the recombinant polypeptide. For example, anydomain of a recombinant polypeptide as disclosed herein can be locatedN-terminal to any one or more other domains in the recombinantpolypeptide. Similarly, any domain of a recombinant polypeptide asdisclosed here can be located C-terminal to any one or more otherdomains in the recombinant polypeptide.

The present disclosure is directed to a recombinant polypeptidecomprising any two or more domains selected from the group consistingSEQ ID NOs: 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, and 355 that has a higher affinity forthe extracellular domain of activin receptor IIB protein (i.e.,ActRIIBecd) than any of the individual domains in the recombinantpolypeptide. Nucleic acid sequences and polypeptide sequences of ActRIIBand naturally occurring variants are known. For example, ActRIIBecd canbe SEQ ID NO: 9 as disclosed herein, which corresponds to residues27-117 of SEQ ID NO: 8 as disclosed herein. Affinity can be as measured,e.g., by a radioimmunoassay (RIA), surface plasmon resonance, such asBIAcore™, or any other binding assay known in the art. In someembodiments, such recombinant polypeptides include the combination oftwo domains, wherein either of the two domains is located N-terminal orC-terminal to the other domain, selected from the following combinationsof domains: SEQ ID NO: 39 and SEQ ID NO: 49, SEQ ID NO: 49 and SEQ IDNO: 61, SEQ ID NO: 61 and SEQ ID NO:39, SEQ ID NO: 35 and SEQ ID NO: 47,SEQ ID NO: 57 and SEQ ID NO: 35, and SEQ ID NO: 57 and SEQ ID NO: 47. Insome embodiments, such combination of two domains yields a recombinantpolypeptide comprising a sequence selected from the group consisting of:SEQ ID NOs: 188, 194, 200, 206, 212, 218, 224, 230, 236, 242, 248, and254. In some embodiments, such recombinant polypeptides include thecombination of three domains, wherein any domain is located N-terminalor C-terminal to one or both of the other domains, selected from thefollowing combinations of domains: SEQ ID NOs: 39, 49, and 61; SEQ IDNOs: 35, 47, and 57; SEQ ID NOs: 39, 47, and 61; SEQ ID NOs: 35, 49, and57; SEQ ID NOs: 39, 57, and 47; and SEQ ID NOs: 35, 61, and 49. In someembodiments, such combination of three domains yields a recombinantpolypeptide comprising a sequence selected from the group consisting of:SEQ ID NOs: 260, 268, 276, 284, 292, 300, 308, 316, 324, 332, 340, and348.

The present disclosure is directed to a recombinant polypeptidecomprising a first domain of SEQ ID NO: 39, a second domain of SEQ IDNO: 49, and a third domain of SEQ ID NO: 61, wherein the first domain islocated C-terminal to the second domain, the third domain is locatedN-terminal to the second domain, or a combination thereof. In certainembodiments, the recombinant polypeptide comprises a first domainselected from the group consisting of SEQ ID NO: 35 and SEQ ID NO: 39, asecond domain selected from the group consisting of SEQ ID NO: 47 andSEQ ID NO: 49, and a third domain selected from the group consisting ofSEQ ID NO: 57 and SEQ ID NO: 61, wherein the first domain is locatedC-terminal to the second domain, the third domain is located N-terminalto the second domain, or a combination thereof when the first, second,and third domains are SEQ ID NOs: 39, 49, and 61, respectively.

In certain embodiments, a recombinant polypeptide as described hereincomprises a post-translation modification (“PTM”), including withoutlimitation disulfide bond formation, glycosylation, carbamylation,lipidation, acetylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, modification bynon-naturally occurring amino acids, or any other manipulation ormodification, such as conjugation with a labeling component.

In certain embodiments, a recombinant polypeptide can include one ormore cysteines capable of participating in formation of one or moredisulfide bonds under physiological conditions or any other standardcondition (e.g., purification conditions or storage conditions). Incertain embodiments, a disulfide bond is an intramolecular disulfidebond formed between two cysteine residues in the recombinantpolypeptide. In certain embodiments, a disulfide bond is anintermolecular disulfide bond formed between two recombinantpolypeptides in a dimer. In certain embodiments, an intermoleculardisulfide bond is formed between two identical recombinant polypeptidesas described herein, wherein the two identical recombinant polypeptidesform a homodimer. In certain embodiments, a homodimer includes at leastone or more than three intermolecular disulfide bonds. In certainembodiments, an intermolecular disulfide bond is formed between twodifferent recombinant polypeptides as described herein, wherein the twodifferent recombinant polypeptides form a heterodimer. In certainembodiments, a heterodimer includes at least one or more than threeintermolecular disulfide bonds.

The present disclosure is directed to a recombinant polypeptidecomprising a first domain selected from the group consisting of SEQ IDNO: 35 and SEQ ID NO: 39, a second domain selected from the groupconsisting of SEQ ID NO: 47 and SEQ ID NO: 49, and a third domainselected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 61,wherein the recombinant polypeptide comprises an intramoleculardisulfide bond.

In certain embodiments, the first domain, second domain, third domain,or combinations thereof comprise an intramolecular disulfide bond. Incertain embodiments, one or more intramolecular disulfide bonds arewithin a single domain, are between one domain and another domain, arebetween one domain with more than two cysteines and one or more otherdomains, or a combination thereof. In certain embodiments, the firstdomain comprises a disulfide bond. In certain embodiments, the seconddomain comprises a disulfide bond. In certain embodiments, the thirddomain comprises a disulfide bond. In certain embodiments, each domaincomprises a disulfide bond. “Domain comprises a disulfide bond” as usedherein when referring to an intramolecular disulfide bond refers to adisulfide bond between two cysteines in a single domain when more thanone cysteine is present in a domain or between a cysteine in one domainand a cysteine in another domain.

In certain embodiments, the second domain of a recombinant polypeptideas described herein comprises an intramolecular disulfide bond betweenthe twenty-third amino acid of the second domain and the twenty-seventhamino acid of the second domain. In certain embodiments, the recombinantpolypeptide further comprises one or more additional intramoleculardisulfide bonds between the first domain and the third domain, withinthe third domain, or both. In certain embodiments, the recombinantpolypeptide further comprises an intramolecular disulfide bond betweenthe ninth amino acid of the third domain and the forty-third amino acidof the third domain, between the eighth amino acid of the third domainand the forty-first amino acid of the third domain, between the eighthamino acid of the third domain and the forty-third amino acid of thethird domain, or between the ninth amino acid of the third domain andthe forty-first amino acid of the third domain. In certain embodiments,the recombinant polypeptide further comprises a disulfide bond betweenthe ninth amino acid of the third domain and the forty-third amino acidof the third domain, and a disulfide bond between the eighth amino acidof the third domain and the forty-first amino acid of the third domain.In certain embodiments, the recombinant polypeptide further comprises adisulfide bond between the eighth amino acid of the third domain and theforty-third amino acid of the third domain, and a disulfide bond betweenthe ninth amino acid of the third domain and the forty-first amino acidof the third domain.

In certain embodiments, the third domain of a recombinant polypeptide asdescribed herein comprises a first amino acid sequence of PKACCVPTE (SEQID NO: 356) and a second amino acid sequence of GCGCR (SEQ ID NO: 357),wherein the third domain comprises either two intramolecular disulfidebonds or two intermolecular disulfide bonds between the first and secondamino acid sequences. In certain embodiments, the recombinantpolypeptide comprises a first intramolecular or intermolecular disulfidebond between the fourth amino acid of the first amino acid sequence andthe second amino acid of the second amino acid sequence, and a secondintramolecular or intermolecular disulfide bond between the fifth aminoacid of the first amino acid sequence and the fourth amino acid of thesecond amino acid sequence. In certain embodiments, the recombinantpolypeptide comprises a first intramolecular or intermolecular disulfidebond between the fifth amino acid of the first amino acid sequence andthe second amino acid of the second amino acid sequence, and a secondintramolecular or intermolecular disulfide bond between the fourth aminoacid of the first amino acid sequence and the fourth amino acid of thesecond amino acid sequence. In certain embodiments, the recombinantpolypeptide further comprises an intramolecular disulfide bond betweenthe twenty-third amino acid of the second domain and the twenty-seventhamino acid of the second domain.

The present disclosure is directed to a recombinant polypeptidecomprising an amino acid sequence selected from the group consisting of:SEQ ID NO: 260, SEQ ID NO: 268, SEQ ID NO: 276, SEQ ID NO: 284, SEQ IDNO: 292, SEQ ID NO: 300, SEQ ID NO: 308, SEQ ID NO: 316, SEQ ID NO: 324,SEQ ID NO: 332, SEQ ID NO: 340, and SEQ ID NO: 348, wherein therecombinant polypeptide comprises an intramolecular disulfide bond. Incertain embodiments, the intramolecular disulfide bond comprises one ormore disulfide bonds comprising cysteine 15, cysteine 44, cysteine 48,cysteine 79, cysteine 80, cysteine 112, cysteine 114, and combinationsthereof, as numbered from the amino-terminus of a recombinantpolypeptide selected from the group consisting of: SEQ ID NO: 260, SEQID NO: 292, SEQ ID NO: 324, and SEQ ID NO: 332. In certain embodiments,the intramolecular disulfide bond comprises cysteine 44, cysteine 48, orboth as numbered from the amino-terminus of a recombinant polypeptideselected from the group consisting of: SEQ ID NO: 260, SEQ ID NO:292,SEQ ID NO: 324, and SEQ ID NO: 332.

In certain embodiments, a recombinant polypeptide selected from thegroup consisting of: SEQ ID NO: 260, SEQ ID NO: 292, SEQ ID NO: 324, andSEQ ID NO: 332 comprises an intramolecular disulfide bond betweencysteine 44 and cysteine 48, as numbered from the amino-terminus of therecombinant polypeptide. In certain embodiments, the recombinantpolypeptide further comprises either an intramolecular or intermolecular(i.e., in a dimer) disulfide bond between cysteine 79 and cysteine 112,between cysteine 80 and cysteine 114, between cysteine 80 and cysteine112, or between cysteine 79 and cysteine 114. In certain embodiments,the recombinant polypeptide further comprises either an intramolecularor intermolecular disulfide bond between cysteine 79 and cysteine 112,and either an intramolecular or intermolecular disulfide bond betweencysteine 80 and cysteine 114. In certain embodiments, the recombinantpolypeptide further comprises either an intramolecular or intermoleculardisulfide bond between cysteine 80 and cysteine 112, and either anintramolecular or intermolecular disulfide bond between cysteine 79 andcysteine 114.

The present disclosure is directed to a homodimeric protein comprisingtwo identical recombinant polypeptides as described herein.

The present disclosure is directed to a heterodimeric protein comprisingtwo different recombinant polypeptides as described herein.

In certain embodiments, a homodimeric protein or heterodimeric proteinas described herein comprises one or more intermolecular disulfide bondsbetween the first domains of the two recombinant polypeptides, betweenthe second domains of the two recombinant polypeptides, between thethird domains of the two recombinant polypeptides, between the first andsecond domains of the two recombinant polypeptides, between the firstand third domains of the two recombinant polypeptides, between thesecond and third domains of the two recombinant polypeptides, orcombinations thereof.

In certain embodiments, a homodimeric or heterodimeric protein asdescribed herein comprises an intermolecular disulfide bond between thefifteenth amino acid of the first domain of one recombinant polypeptideand the fifteenth amino acid of the first domain of the otherrecombinant polypeptide. In certain embodiments, the homodimeric orheterodimeric protein further comprises an intermolecular disulfide bondbetween the ninth amino acid of the third domain of one recombinantpolypeptide and the forty-third amino acid of the third domain of theother recombinant polypeptide, between the eighth amino acid of thethird domain of one recombinant polypeptide and the forty-first aminoacid of the third domain of the other recombinant polypeptide, betweenthe eighth amino acid of the third domain of one recombinant polypeptideand the forty-third amino acid of the third domain of the otherrecombinant polypeptide, or between the ninth amino acid of the thirddomain of one recombinant polypeptide and the forty-first amino acid ofthe third domain of the other recombinant polypeptide. In certainembodiments, the homodimeric or heterodimeric protein further comprisesa disulfide bond between the ninth amino acid of the third domain of onerecombinant polypeptide and the forty-third amino acid of the thirddomain of the other recombinant polypeptide, and a disulfide bondbetween the eighth amino acid of the third domain of one recombinantpolypeptide and the forty-first amino acid of the third domain of theother recombinant polypeptide. In certain embodiments, the homodimericor heterodimeric protein further comprises a disulfide bond between theeighth amino acid of the third domain of one recombinant polypeptide andthe forty-third amino acid of the third domain of the other recombinantpolypeptide, and a disulfide bond between the ninth amino acid of thethird domain of one recombinant polypeptide and the forty-first aminoacid of the third domain of the other recombinant polypeptide.

In certain embodiments, the homodimeric or heterodimeric proteincomprises an intermolecular disulfide bond between the ninth amino acidof the third domain of one recombinant polypeptide and the forty-thirdamino acid of the third domain of the other recombinant polypeptide,between the eighth amino acid of the third domain of one recombinantpolypeptide and the forty-first amino acid of the third domain of theother recombinant polypeptide, between the eighth amino acid of thethird domain of one recombinant polypeptide and the forty-third aminoacid of the third domain of the other recombinant polypeptide, orbetween the ninth amino acid of the third domain of one recombinantpolypeptide and the forty-first amino acid of the third domain of theother recombinant polypeptide. In certain embodiments, the homodimericor heterodimeric protein comprises a disulfide bond between the ninthamino acid of the third domain of one recombinant polypeptide and theforty-third amino acid of the third domain of the other recombinantpolypeptide, and a disulfide bond between the eighth amino acid of thethird domain of one recombinant polypeptide and the forty-first aminoacid of the third domain of the other recombinant polypeptide. Incertain embodiments, the homodimeric or heterodimeric protein comprisesa disulfide bond between the eighth amino acid of the third domain ofone recombinant polypeptide and the forty-third amino acid of the thirddomain of the other recombinant polypeptide, and a disulfide bondbetween the ninth amino acid of the third domain of one recombinantpolypeptide and the forty-first amino acid of the third domain of theother recombinant polypeptide.

In certain embodiments, the third domain of each recombinant polypeptideof a homodimeric or heterodimeric protein as described herein comprisesa first amino acid sequence of PKACCVPTE (SEQ ID NO: 356) and a secondamino acid sequence of GCGCR (SEQ ID NO: 357), wherein the homodimericor heterodimeric protein comprises two intermolecular disulfide bondsbetween the first amino acid sequence in the third domain of onerecombinant polypeptide and the second amino acid sequence in the thirddomain of the other recombinant polypeptide. In certain embodiments, thehomodimeric or heterodimeric protein comprises a first intermoleculardisulfide bond between the fourth amino acid of the first amino acidsequence of the one recombinant polypeptide and the second amino acid ofthe second amino acid sequence of the other recombinant polypeptide, anda second intermolecular disulfide bond between the fifth amino acid ofthe first amino acid sequence of the one recombinant polypeptide and thefourth amino acid of the second amino acid sequence of the otherrecombinant polypeptide. In certain embodiments, the homodimeric orheterodimeric protein comprises a first intermolecular disulfide bondbetween the fifth amino acid of the first amino acid sequence of the onerecombinant polypeptide and the second amino acid of the second aminoacid sequence of the other recombinant polypeptide, and a secondintermolecular disulfide bond between the fourth amino acid of the firstamino acid sequence of the one recombinant polypeptide and the fourthamino acid of the second amino acid sequence of the other recombinantpolypeptide.

In certain embodiments, one or both of the recombinant polypeptides of ahomodimeric or heterodimeric protein as described herein comprises anyone or more of the intramolecular disulfide bonds as described herein.

In certain embodiments, the second domain of one or both of therecombinant polypeptides of a homodimeric or heterodimeric protein asdescribed herein comprises an intramolecular disulfide bond. In certainembodiments, one or both of the recombinant polypeptides of ahomodimeric or heterodimeric protein as described herein comprises anintramolecular disulfide bond between the twenty-third amino acid of thesecond domain and the twenty-seventh amino acid of the second domain.

In certain embodiments, one or both of the recombinant polypeptides of ahomodimeric or heterodimeric protein as described herein comprises anintramolecular disulfide bond between cysteine 44 and cysteine 48 forany of SEQ ID NOs: 260, 292, 324, or 332, between cysteine 88 andcysteine 92 for any of SEQ ID NOs: 284, 308, 340, 348, between cysteine23 and cysteine 27 for SEQ ID NOs: 268 or 300, or between cysteine 67and cysteine 71 for SEQ ID NOs: 276 or 316, as numbered from theamino-terminus of the recombinant polypeptide.

In certain embodiments, a homodimeric protein as described hereincomprises two recombinant polypeptides, wherein each polypeptidecomprises the same sequence, and wherein the sequence is selected fromthe group consisting of: SEQ ID NO: 260, SEQ ID NO: 268, SEQ ID NO: 276,SEQ ID NO: 284, SEQ ID NO: 292, SEQ ID NO: 300, SEQ ID NO: 308, SEQ IDNO: 316, SEQ ID NO: 324, SEQ ID NO: 332, SEQ ID NO: 340, and SEQ ID NO:348. In some embodiments, the recombinant polypeptides compriseidentical intramolecular disulfide bonds as described herein. In someembodiments, the recombinant polypeptides comprise differentintramolecular disulfide bonds as described herein.

In certain embodiments, a heterodimeric protein as described hereincomprises two different recombinant polypeptides, wherein eachpolypeptide comprises a different sequence selected from the groupconsisting of: SEQ ID NO: 260, SEQ ID NO: 268, SEQ ID NO: 276, SEQ IDNO: 284, SEQ ID NO: 292, SEQ ID NO: 300, SEQ ID NO: 308, SEQ ID NO: 316,SEQ ID NO: 324, SEQ ID NO: 332, SEQ ID NO: 340, and SEQ ID NO: 348. Incertain embodiments, one recombinant polypeptide in the heterodimericprotein comprises the sequence of SEQ ID NO: 260 and the otherrecombinant polypeptide comprises a sequence selected from the groupconsisting of: SEQ ID NO: 268, SEQ ID NO: 276, SEQ ID NO: 284, SEQ IDNO: 292, SEQ ID NO: 300, SEQ ID NO: 308, SEQ ID NO: 316, SEQ ID NO: 324,SEQ ID NO: 332, SEQ ID NO: 340, and SEQ ID NO: 348. In some embodiments,the recombinant polypeptides comprise identical intramolecular disulfidebonds as described herein. In some embodiments, the recombinantpolypeptides comprise different intramolecular disulfide bonds asdescribed herein.

In certain embodiments, a recombinant polypeptide, homodimeric protein,or heterodimeric protein as described herein comprises one or more ofthe disulfide bonds between cysteine pairs as listed in Table 4 or Table5 herein.

In certain embodiments, a recombinant polypeptide, homodimeric protein,or heterodimeric protein as described herein comprises an osteoinductiveactivity. Osteoinductive activity can be measured under any conditionsroutinely practiced for measuring such activity (i.e., “osteoinductiveconditions”).

For example, C2C12 cells are a murine myoblast cell line from dystrophicmouse muscle. Exposure of C2C12 cells to a polypeptide withosteoinductive activity can shift C2C12 cell differentiation from muscleto bone, for example, by inducing osteoblast formation characterized byexpression of a bone-associated protein such as alkaline phosphatase.Alkaline phosphatase is a widely accepted bone marker, and assays fordetection of alkaline phosphatase activity are accepted as demonstratingosteoinductive activity. See, e.g., Peel et al., J. Craniofacial Surg.14: 284-291 (2003); Hu et al., Growth Factors 22: 29033 (2004); and Kimet al., J. Biol. Chem. 279: 50773-50780 (2004).

In certain embodiments, a recombinant polypeptide, homodimeric protein,or heterodimeric protein as described herein is capable of inducingalkaline phosphatase activity.

In certain embodiments, osteoinductive activity is detected by a medicalimaging technology or histological examination of bone samples, or anyother method routinely practiced for detection of bone formation orgrowth. In certain embodiments, the detection comprises radiographicimaging, such as X-ray imaging. In certain embodiments, the detectioncomprises computed tomography (CT) scanning. In some embodiments, thedetection comprises molecular imaging or nuclear imaging (i.e., positronemission tomography (PET)). In certain embodiments, the detectioncomprises histological examination. In certain embodiments, thedetection comprises hematoxylin and eosin (HE)-staining.

In certain embodiments, a recombinant polypeptide, homodimeric protein,or heterodimeric protein as described herein can include fragment,variant, or derivative molecules thereof without limitation. The terms“fragment,” “variant,” “derivative” and “analog” when referring to apolypeptide include any polypeptide which retains at least some propertyor biological activity of the reference polypeptide. Polypeptidefragments can include proteolytic fragments, deletion fragments, andfragments which more easily reach the site of action when implanted inan animal. Polypeptide fragments can comprise variant regions, includingfragments as described above, and also polypeptides with altered aminoacid sequences due to amino acid substitutions, deletions, orinsertions. Non-naturally occurring variants can be produced usingart-known mutagenesis techniques. Polypeptide fragments of thedisclosure can comprise conservative or non-conservative amino acidsubstitutions, deletions, or additions. Variant polypeptides can also bereferred to herein as “polypeptide analogs.” Polypeptide fragments ofthe present disclosure can also include derivative molecules. As usedherein a “derivative” of a polypeptide or a polypeptide fragment refersto a subject polypeptide having one or more residues chemicallyderivatized by reaction of a functional side group. Also included as“derivatives” are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexample, 4-hydroxyproline can be substituted for proline;5-hydroxylysine can be substituted for lysine; 3-methylhistidine can besubstituted for histidine; homoserine can be substituted for serine; andornithine can be substituted for lysine.

In certain embodiments, a recombinant polypeptide, homodimeric protein,or heterodimeric protein as described herein comprises a label. Incertain embodiments, the label is an enzymatic label that can catalyzethe chemical alteration of a substrate compound or composition, aradiolabel, a fluorophore, a chromophore, an imaging agent, or a metal,including a metal ion.

In certain embodiments, a recombinant polypeptide as described hereincomprises one or more conservative amino acid substitutions. A“conservative amino acid substitution” is a substitution of an aminoacid with different amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, if an amino acid in apolypeptide is replaced with another amino acid from the same side chainfamily, the substitution is considered to be conservative. In anotherembodiment, a string of amino acids can be conservatively replaced witha structurally similar string that differs in order and/or compositionof side chain family members.

In certain embodiments, a recombinant polypeptide of the disclosure isencoded by a nucleic acid molecule or vector of the disclosure asdescribed herein, or is expressed by a host cell as described herein.

Nucleic Acid Molecules, Vectors, and Host Cells

The present disclosure is directed to an isolated nucleic acid moleculecomprising a polynucleotide sequence encoding any of the recombinantpolypeptides described herein.

In certain embodiments, the isolated nucleic acid molecule comprises anytwo or more polynucleotide sequences encoding a domain as describedherein. In certain embodiments, the isolated nucleic acid moleculecomprises any two or more polynucleotide sequences selected from thegroup consisting of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, which encodedomains described herein corresponding to SEQ ID NOs: 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, and 355, respectively. In certain embodiments, the isolated nucleicacid molecule comprises any combination of two or three polynucleotidesequences encoding the corresponding combinations of two or threedomains shown in Table 3, herein.

In certain embodiments, the isolated nucleic acid molecule comprises apolynucleotide sequence selected from the group consisting of SEQ IDNOs: 115, 157, 187, 193, 199, 205, 211, 217, 223, 229, 235, 241, 247,253, 259, 267, 275, 283, 291, 299, 307, 315, 323, 331, 339, and 347,which encodes a recombinant polypeptide described herein correspondingto SEQ ID NOs: 116, 158, 188, 194, 200, 206, 212, 218, 224, 230, 236,242, 248, 254, 260, 268, 276, 284, 292, 300, 308, 316, 324, 332, 340,and 348, respectively.

In certain embodiments, the polynucleotide sequence is codon-optimized.

The present disclosure is directed to a recombinant nucleic acidmolecule comprising an expression control region operably linked to anisolated nucleic acid molecule as described herein. In certainembodiments, the expression control region is a promoter, enhancer,operator, repressor, ribosome binding site, translation leader sequence,intron, polyadenylation recognition sequence, RNA processing site,effector binding site, stem-loop structure, transcription terminationsignal, or combination thereof. In certain embodiments, the expressioncontrol region is a promoter. An expression control region can be atranscription control region and/or a translation control region.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions, which function in vertebrate cells, such as, but not limitedto, promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit ß-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In certain embodiments, the recombinant nucleic acid molecule is arecombinant vector.

A vector can be any vehicle for the cloning of and/or transfer of anucleic acid into a host cell. A large number of vectors are known andused in the art including, for example, plasmids, phages, cosmids,chromosomes, viruses, modified eukaryotic viruses, modified bacterialviruses. Insertion of a polynucleotide into a suitable vector can beaccomplished by ligating the appropriate polynucleotide fragments into achosen vector that has complementary cohesive termini.

Vectors can be engineered to encode selectable markers or reporters thatprovide for the selection or identification of cells that haveincorporated the vector. Expression of selectable markers or reportersallows identification and/or selection of host cells that incorporateand express other coding regions contained on the vector. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, neomycin, puromycin, bialaphos herbicide, sulfonamide, andthe like; and genes that are used as phenotypic markers, i.e.,anthocyanin regulatory genes, isopentanyl transferase gene, and thelike. Examples of reporters known and used in the art include:luciferase (Luc), green fluorescent protein (GFP), chloramphenicolacetyltransferase (CAT), beta-galactosidase (LacZ), beta-glucuronidase(Gus), and the like. Selectable markers may also be considered to bereporters.

The term “plasmid” refers to an extra-chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements can be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

Eukaryotic viral vectors that can be used include, but are not limitedto, adenovirus vectors, retrovirus vectors, adeno-associated virusvectors, and poxvirus, e.g., vaccinia virus vectors, baculovirusvectors, or herpesvirus vectors. Non-viral vectors include plasmids,liposomes, electrically charged lipids (cytofectins), DNA-proteincomplexes, and biopolymers. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences.

The recombinant vector can be a “recombinant expression vector,” whichrefers to any nucleic acid construct which contains the necessaryelements for the transcription and translation of an inserted codingsequence, or in the case of an RNA viral vector, the necessary elementsfor replication and translation, when introduced into an appropriatehost cell.

The present disclosure is directed to a method of making a recombinantvector comprising inserting an isolated nucleic acid molecule asdescribed herein into a vector.

The present disclosure is directed to an isolated host cell comprisingan isolated nucleic acid molecule or recombinant nucleic acid moleculeas described herein. In certain embodiments, the isolated host cellcomprises a recombinant vector as described herein.

Nucleic acid molecules can be introduced into host cells by methods wellknown in the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter.

The present disclosure is directed to a method of making a recombinanthost cell comprising introducing an isolated nucleic acid molecule orrecombinant nucleic acid molecule as described herein into a host cell.In certain embodiments, the method comprises introducing a recombinantvector as described herein into a host cell.

A host cell as described herein can express any of the isolated nucleicacid molecules or recombinant nucleic acid molecules described herein.The term “express” as used with respect to expression of a nucleic acidmolecule in a host cell refers to a process by which a gene produces abiochemical, for example, a RNA or polypeptide. The process includes anymanifestation of the functional presence of the gene within the cellincluding, without limitation, transient expression or stableexpression. It includes, without limitation, transcription of the geneinto messenger RNA (mRNA), and the translation of such mRNA intopolypeptide(s).

Host cells include, without limitation, prokaryotes or eukaryotes.Representative examples of appropriate host cells include bacterialcells; fungal cells, such as yeast; insect cells; and isolated animalcells. Bacterial cells can include, without limitation, gram negative orgram positive bacteria, for example Escherichia coli. Alternatively, aLactobacillus species or Bacillus species can be used as a host cell.Eukaryotic cells can include, but are not limited to, established celllines of mammalian origin. Examples of suitable mammalian cell linesinclude COS-7, L, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHKcell lines.

The host cells can be cultured in conventional nutrient media modifiedas appropriate for activating promoters, selecting transformants, oramplifying nucleic acid molecules of the present disclosure. The cultureconditions, such as temperature, pH, and the like, can be any conditionsknown to be used or routinely modified when using the host cell selectedfor expression, and will be apparent to the ordinarily skilled artisan.

The present disclosure is directed to a method of producing arecombinant polypeptide, comprising: culturing an isolated host cell asdescribed herein and isolating the recombinant polypeptide from the hostcell. Techniques for isolating polypeptides from cultured host cells canbe any technique known to be used or routinely modified when isolatingpolypeptides from the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Compositions and Devices

The present disclosure is directed to a composition comprising arecombinant polypeptide, homodimeric protein, or heterodimeric proteinas described herein.

In certain embodiments, the composition further comprises aphysiologically acceptable carrier, excipient, or stabilizer. See, e.g.,Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton,Pa. Acceptable carriers, excipients, or stabilizers can include thosethat are nontoxic to a subject. In certain embodiments, the compositionor one or more components of the composition are sterile. A sterilecomponent can be prepared, for example, by filtration (e.g., by asterile filtration membrane) or by irradiation (e.g., by gammairradiation).

In certain embodiments, a composition as described herein furthercomprises an allograft or autograft of bone or bone fragments.

In certain embodiments, a composition as described herein furthercomprises a bone graft substitute.

In certain embodiments, the bone graft substitute is a bioceramicmaterial. The terms “bioceramic material” and “bioceramic” can be usedinterchangeably herein. In certain embodiments, the bioceramic isbiocompatible and is resorbable in vivo. In certain embodiments, thebioceramic is any calcium phosphate salt-based bioceramic. In certainembodiments, the bioceramic is selected from the group consisting oftricalcium phosphate (TCP), alpha-tricalcium phosphate (α-TCP),beta-tricalcium phosphate (β-TCP), biphasic calcium phosphate (BCP),hydroxylapatite, calcium sulfate, and calcium carbonate. In certainembodiments, the bioceramic is beta-tricalcium phosphate (β-TCP).

In certain embodiments, the bone graft substitute is a bioactive glass.In certain embodiments, the bioactive glass comprises silicon dioxide(SiO₂), sodium oxide (Na₂O), calcium oxide (CaO), or platinum oxide(Pt₂O₅).

The present disclosure is directed to a biodegradable composition,comprising: a homodimeric protein as disclosed herein, being able toinduce bone growth to form a bone mass in a location; and abiodegradable calcium phosphate carrier (e.g., β-TCP) with pores thatextend throughout the biodegradable calcium phosphate carrier, whereinthe homodimeric protein is in an effective amount of from about 0.03mg/g to about 3.2 mg/g of the biodegradable calcium phosphate carrierand porosity of the biodegradable calcium phosphate carrier is more than70% with pore size from about 300 μm to about 600 μm.

In certain embodiments, the biodegradable composition is suitable foraugmentation of a tissue selected from the group consisting of: nasalfurrows, frown lines, midfacial tissue, jaw-line, chin, cheeks, andcombinations thereof.

In certain embodiments, the location is selected from the groupconsisting of: a long-bone fracture defect, a space between two adjacentvertebra bodies, a non-union bone defect, maxilla osteotomy incision,mandible osteotomy incision, sagittal split osteotomy incision,genioplasty osteotomy incision, rapid palatal expansion osteotomyincision, a space extending lengthwise between two adjacent transverseprocesses of two adjacent vertebrae, and combinations thereof.

In certain embodiments, a single dose of the homodimeric protein is fromabout 0.006 mg to about 15 mg.

In certain embodiments, the biodegradable calcium phosphate carrierhardens so as to be impermeable to efflux of the homodimeric protein invivo sufficiently that the formed bone mass is confined to the volume ofthe biodegradable calcium phosphate carrier.

The present disclosure is directed to a sustained release composition,comprising a calcium phosphate carrier, a biodegradable matrix, and ahomodimeric protein as disclosed herein.

In certain embodiments, the calcium phosphate carrier is selected fromthe group consisting of: tricalcium phosphate (TCP), alpha-tricalciumphosphate (α-TCP), beta-tricalcium phosphate (β-TCP), biphasic calciumphosphate (BCP), and combinations thereof.

In certain embodiments, the biodegradable matrix is selected from thegroup consisting of: polylactic acid (PLA), polyglycolic acid (PGA),polylactic-co-glycolic acid (PLGA), Polyvinyl alcohol (PVA), andcombinations thereof.

In certain embodiments, the sustained release composition comprises: (a)about 2-11% (w/w) of the calcium phosphate carrier; (b) about 88-97%(w/w) of the biodegradable matrix; and (c) about 0.017-0.039% (w/w) ofthe homodimeric protein.

The present disclosure is directed to a moldable composition for fillingan osseous void, comprising: a moldable matrix comprising about 90% toabout 99.5% by weight of the moldable composition; and a homodimericprotein as disclosed herein, wherein less than about 25% by percentageof the homodimeric protein is released from the moldable compositionafter about 1, 24, 48, 72, 168, 240 or about 336 hours postimplantation.

The present disclosure is directed to a spinal fusion device comprisinga biodegradable composition as disclosed herein; and a spinal fusioncage (e.g., peek cage), configured to retain the biodegradable calciumphosphate carrier.

Methods

The present disclosure is directed to a method of promoting healing of along-bone fracture in a subject in need of such treatment, comprising:preparing a composition including a homodimeric protein as disclosedherein homogeneously entrained within a slow release biodegradablecalcium phosphate carrier (e.g., β-TCP) that hardens so as to beimpermeable to efflux of the homodimeric protein in vivo sufficientlythat the long-bone fracture healing is confined to the volume of thecalcium phosphate carrier; and implanting the composition at a locationwhere the long-bone fracture occurs, wherein the homodimeric protein isin an amount of from about 0.03 mg/g to about 3.2 mg/g of the calciumphosphate carrier.

In certain embodiments, the method of promoting healing of a long-bonefracture further comprises gradually exposing the entrained homodimericprotein at the location as the calcium phosphate carrier degrades,wherein the calcium phosphate carrier has a calcium to phosphate ratioof about 0.4 to about 1.8.

The present disclosure is directed to a method for promoting spinalfusion, comprising: exposing an upper vertebra and a lower vertebra;identifying a site for fusion between the upper and the lower vertebra;exposing a bone surface on each of the upper and the lower vertebra atthe site for fusion; and administering a homodimeric protein asdisclosed herein and a biodegradable calcium phosphate carrier (e.g.,β-TCP) to the site.

In certain embodiments, the biodegradable calcium phosphate carrier is anon-compressible delivery vehicle, and wherein the non-compressibledelivery vehicle is for application to the site between the two bonesurfaces where bone growth is desired but does not naturally occur.

In certain embodiments, the biodegradable calcium phosphate carriercomprises at least one implantation stick for application to the sitesuch that the implantation stick extends lengthwise between the upperand the lower vertebrae.

In certain embodiments, the site is selected from a space between twoadjacent vertebra bodies, and a space extending lengthwise between twoadjacent transverse processes of two adjacent vertebrae.

The present disclosure is directed to a method for promotingarthrodesis, comprising administering a homodimeric protein as disclosedherein and a biodegradable calcium phosphate carrier to a malformed ordegenerated joint.

In certain embodiments, administering the homodimeric protein includesadministering from about 0.006 mg to about 10.5 mg of the homodimericprotein to the malformed or degenerated joint.

The present disclosure is directed to a method of generating a bone massto fuse two adjacent vertebrae bodies in a spine of a subject in need,comprising: preparing a composition for generating the bone mass, thecomposition including a homodimeric protein as disclosed hereinhomogeneously entrained within a slow release biodegradable carrier thathardens so as to be impermeable to efflux of the homodimeric protein invivo sufficiently that the formed bone mass is confined to the volume ofthe slow release biodegradable carrier; and introducing the compositionin a location between the two adjacent vertebrae bodies, and wherein theslow release biodegradable carrier gradually exposes the entrainedhomodimeric protein at the location as the slow release biodegradablecarrier degrades, and further wherein the homodimeric protein is in anamount of from about 0.2 mg/site to about 10.5 mg/site of the location.

In certain embodiments, the slow release biodegradable carrier has aporous structure, wherein cells from the two adjacent vertebrae migrateinto the porous structure so as to generate the bone mass.

In certain embodiments, the slow release biodegradable carrier has aninitial volume, and the bone mass replaces the initial volume of theslow release biodegradable carrier as the slow release biodegradablecarrier is resorbed.

The present disclosure is directed to a method for fusing adjacentvertebrae bodies in a subject in need by a posterior or transforaminalfusion approach, comprising: preparing a disc space for receipt of anintervertebral disc implant in an intervertebral space between theadjacent vertebrae; introducing a slow-release carrier into theintervertebral disc implant, wherein a homodimeric protein as disclosedherein is in an amount of from about 0.2 mg/site to about 10.5 mg/siteof the slow-release carrier; and introducing the intervertebral discimplant in the disc space between the adjacent vertebrae for generatinga bone mass in the disc space.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1: Plasmid Construction

For construction of plasmid pQE-80L-Kana, Kanamycin resistance gene wascleaved from pET-24a(+) (Novagen) by BspHI (BioLab) to generate a 875-bpKanamycin resistance gene (+3886 to +4760) fragment (SEQ ID NO: 1). ThepQE-80L (Qiagen) vector was digested with BspHI to remove Ampicillinresistance gene (+3587 to +4699) fragment (SEQ ID NO:2) and then theKanamycin resistance gene fragment was ligated into the pQE-80L vectorto generate a 4513-bp plasmid (pQE-80L-Kana). (SEQ ID NO:3)

Example 2: Yeast Two-Hybrid Screening

A. Construction of Bait Plasmid

Yeast two-hybrid screening was performed using a commercially availablesystem (Matchmaker Two-Hybrid System 2; CLONTECH, Palo Alto, Calif.). Toconstruct the bait plasmids, the coding region of the extracellulardomain (+103 to +375 bp) (SEQ ID NO: 4) of activin receptor type IIB(ActRIIB) protein was produced by PCR with pCRII/ActRIIB (Hilden, et al.(1994) Blood 83(8):2163-70) as the template. The primers (XmaI:5′-CCCGGGACGGGAGTGCATCTACAACG-3′(SEQ ID NO: 5); SalI:5′-GTCGACTTATGGCAAATGAGTGAAGCGTTC-3′(SEQ ID NO: 6)) used to amplify theextracellular domain of ActRIIB (ActRIIBecd) were designed to include anXmaI and SalI restriction site in the 5′-end, respectively. PCR wascarried out using 10 ng template DNA, 0.2 μM each primer, 0.2 mM eachdNTP, 1×PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl and 1.5 mM MgCl₂),and 1.25 U pfu DNA polymerase (Promega) in a total volume of 50 μl. PCRwas performed with 30 cycles of: 30 seconds of denaturing at 95° C.,followed by annealing at 45° C. for 1 min, and extension at 68° C. for 5min. The PCR products were digested with XmaI-SalI and then subcloned inframe into the same restriction sites in the DNA-binding domain of GAL4in the pAS2-1 vector (CLONTECH, GenBank Accession No.: U30497) togenerate plasmid pAS-ActRIIBecd.

The nucleic acid sequences and polypeptide sequences of ActRIIB andnaturally occurring variants are known. For example, a wild-type ActRIIBnucleic acid sequence is SEQ ID NO: 7. The corresponding polypeptidesequence is SEQ ID NO: 8. The extracellular domain of ActRIIB(ActRIIBecd) is SEQ ID NO: 9, which corresponds to residues 27-117 ofSEQ ID NO: 8 and is encoded by the nucleic acid sequence of SEQ ID NO:4.

B. Construction of pACT2/MC3T3 cDNA Library

To construct pACT2/MC3T3 cDNA library, approximately 7×10⁶ clones of amouse MC3T3-E1 osteoblast cDNA library described by Tu Q., et al. (2003,J Bone Miner Res. 18(10):1825-33), with some modifications to allow cDNAlibrary inserts smaller than 1.5 kb, were constructed into pACT2 vector(CLONTECH, GenBank Accession No.: U29899), wherein after 51 nucleasetreatment (Invitrogen life technologies cDNA Synthesis System CAT. No.18267-013), the double-stranded cDNA was cloned in the pACT2 vector,which had been digested with SmaI to express fusion proteins with theGAL4 activation domain. The pACT2/MC3T3 cDNA library was then screenedby the “HIS3 Jump-Start” procedure according to the protocol from themanufacturer (CLONTECH, Palo Alto, Calif.). In another embodiment, thepACT2 cDNA library is obtained as a commercial product.

C. Selection of Yeast Strain

Saccharomyces cerevisiae Y190 cells (MATa, ura3-52, his3-D200, lys2-801,ade2-101, trp1-901, leu2-3, 112, gal4D, gal80D,URA3::GAL1_(UAS)-GAL1_(TATA)-lacZ, cyh^(r)2,LYS2::GAL_(UAS)-HIS3_(TATA)-HI53; CLONTECH, Palo Alto, Calif.) werefirst transformed with bait plasmids and selected on synthetic dextrosemedium lacking tryptophan (SD-Trp). The transformants grown on theSD-Trp medium were subsequently transformed with the pACT2/MC3T3 cDNAlibrary and selected on medium lacking tryptophan and leucine(SD-Trp-Leu). The clones co-transformed with the bait and library werecollected and replated onto medium lacking tryptophan, leucine andhistidine (SD-Trp-Leu-His) with 30 mM 3-amino-1,2,4-triazole(Sigma-Aldrich, St. Louis, Mo.) to inhibit the leaking growth of Y190cells. The clones selected in this step were further assayed for theirβ-galactosidase activity. Plates were photo-graphed after incubation at30° C. for 3 days. At least three independent experiments wereperformed, with similar results. The pACT2 library plasmids werepurified from individual positive clones and amplified in Escherichiacoli. Sequencing (primer 5′-AATACCACTACAATGGAT-3′ (SEQ ID NO: 10)) ofthe cDNA insert in the positive clones shown in Table 1 was performedwith a Perkin-Elmer ABI Automated DNA Sequencer.

TABLE 1 Clone No./ SEQ ID No. DNA sequence Primers 1/SEQ ID5′-GGCCAAGCCAAACGCAAAGGG 5′-TTAACCATGGGCCAAGCCAAACGC-3′ NO: 11TATAAACGCCTTAAGTCCAAATGT (SEQ ID NO: 12) (The bold font AACATACACCCTTTGTAC-3′ bases is the recognition site ofrestriction endonuclease MseI) 5′-GGATCCTTAGTACAAAGGGTGTATGTTAC-3′(SEQ ID NO: 13) (The bold font  bases is the recognition siteof restriction endonuclease BamHI) 2/SEQ ID 5′-GTGAGCTTCAAAGACATTGGG5′-TTAACCATGGTGAGCTTCAAAGACA-3′ NO: 14 TGGAATGACCATGCTAGCAGCCAG(SEQ ID NO: 15) (The bold font  CCGGGGTATCACGCCCGTCCCTGCbases is the recognition site of CACGGACAATGCCAGAATATTCTGrestriction endonuclease MseI) GCTGATCATCTGAACGAAGATTGT5′-GGATCCTTAAACAGAGCGGGGCTTCAGCT-3′ CATGCCATTGTTCAGCTGAAGCCC(SEQ ID NO: 16) (The bold font  CGCTCTGTT-3′bases is the recognition site of restriction endonuclease BamHI)3/SEQ ID 5′-ATCGTTGTGGATAATAAGGCA 5′-TTAACCATGATCGTTGTGGATAATAAG-3′NO: 17 TGCTGTGTCCCGACAGAACTCAGT (SEQ ID NO: 18) (The bold font CTTCCCCATCCGCTGTACCTTGAC bases is the recognition siteGAGAATAAAAAGCCTGTATATAAG of restriction endonuclease MseI)AACTATCAGGACGCGCTTCTGCAT 5′-GGATCCTTAGCGACACCCACAACTATGCA-3′AGTTGTGGGTGTCGC-3′ (SEQ ID NO: 19) (The bold font bases is the recognition site of restriction endonuclease BamHI)4/SEQ ID 5′-ACGTATCCAGCCTCTCCGAAG 5′-TTAACCATGACGTATCCAGCCTCTCCG-3′NO: 20 CCGATGAGGTGGTCAATGCGGAGC (SEQ ID NO: 21) (The bold font TGCGCGTGCTGCGCCGGAGGTCTC bases is the recognition site ofCGGAACCAGACAGGGACAGTG-3′ restriction endonuclease MseI)5′-GGATCCTTACACTGTCCCTGTCTGGTTCC-3′ (SEQ ID NO: 22) (The bold font bases is the recognition site of restriction endonuclease BamHI)5/SEQ ID 5′-GAGCCCCTGGGCGGCGCGCGC 5′-TTAACCATGGAGCCCCTGGGCGGCGC-3′NO: 23 TGGGAAGCGTTCGACGTGACGGAC (SEQ ID NO: 24) (The bold font GCGGTGCAGAGCCACCGCCGCTGG bases is the recognition site ofCCGCGAGCCTCCCGCAAGTGCTGC restriction endonuclease MseI)CTGGTGCTGCGCGCGGTGACGGCC 5′-GGATCCTTACGAGGCCGTCACCGCGCGCA-3′ TCG-3′(SEQ ID NO: 25) (The bold font  bases is the recognition siteof restriction endonuclease BamHI) 6/SEQ ID 5′-ACTGCGCTGGCTGGGACTCGG5′-TTAACCATGACTGCGCTGGCTGGGAC-3′ NO: 26 GGAGCGCAGGGAAGCGGTGGTGGC(SEQ ID NO: 27) (The bold font  GGCGGTGGCGGTGGCGGCGGCGGCbases is the recognition site of GGCGGCGGCGGCGGCGGCGGCGGCrestriction endonuclease MseI) GGCGCAGGCAGGGGCCACGGGCGC5′-GGATCCTTAGCCCAGCTCCTTAAAGTCCA-3′ AGAGGCCGGAGCCGCTGCAGTCGC(SEQ ID NO: 28) (The bold font  AAGTCACTGCACGTGGACTTTAAGbases is the recognition site GAGCTGGGC-3′of restriction endonuclease BamHI)

Example 3: Error-Prone Random Mutagenic PCR

A. Mutagenization with Primers Designed from Plasmid

The DNA sequences of positive clones from Example 2 were mutagenized.

In one embodiment, sequenced positive clones were subcloned intopQE-80L-Kana and then random mutageneic PCR was performed. The primersin Table 1 used to amplify DNA sequence of positive clones were designedto include a MseI or BamHI restriction site in the 5′-end. The PCRconditions were as described in Example 2. The PCR products weredigested with MseI-BamHI and then subcloned in frame into the samerestriction sites in the pQE-80L-Kana vector. Random mutagenesis wasintroduced into the subcloned pQE-80L-Kana plasmids on the basis of theerror-prone PCR described by Leung et al. (1989, Technique, 1, 11-15),with some modifications. The linearized pQE-80L-Kana (XhoI-digested) wasused as template DNA. The primers (MseI: 5′-GAATTCATTAAAGAGGAGAAATTAA(SEQ ID NO: 29); BamHI: 5′-CCGGGGTACCGAGCTCGCATGCGGATCCTTA (SEQ ID NO:30)) used for mutagenic PCR amplification were designed to include aMseI or BamHI restriction site in the 5′-end, respectively. MutagenicPCR was carried out using 10 ng template DNA, 40 pM each primer, 0.2 mMeach dNTP, 1×PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl and 1.5 mMMgCl₂), 0.2-0.3 mM MnCl₂, 1% dimethyl sulfoxide, and 1.25 U Taq DNApolymerase (Invitrogen, Carlsbad, Calif.) in a total volume of 50 μl.Mutagenic PCR was performed with 30 cycles of: 30 seconds of denaturingat 94° C., followed by annealing at 55° C. for 2 min, and extension at72° C. for 3 min. The PCR product was digested by MseI and BamHI. Thisfragment was ligated with the 4.5-kb fragment of pQE-80L-Kana digestedwith MseI and BamHI. The resulting pQE-80L-Kana derivatives were used totransform E. coli BL21 (Novagen). Colonies were grown on a plate ofLTB-agar medium (LB supplemented with 1% v/v tributyrin, 0.1% v/v Tween80, 100 mg/L of kanamycin, 0.01 μM isopropyl β-D-thiogalactopyranoside,and 1.5% agar) at 37° C.

B. Mutagenization with Primers in TABLE I

In another embodiment, random mutagenesis was introduced into the pACT2library plasmids from the positive clone on the basis of the error-pronePCR previously described by Leung with some modifications. Thelinearized pACT2 (XbaI-digested) was used as template DNA. Syntheticoligonucleotides with MseI and BamHI restriction sites shown in Table 1were used primers for mutagenic PCR amplification. Mutagenic PCR wascarried out using 10 ng template DNA, 40 pM each primer, 0.2 mM eachdNTP, 1×PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl and 1.5 mM MgCl₂),0.2-0.3 mM MnCl₂, 1% dimethyl sulfoxide, and 1.25 U Taq DNA polymerase(Invitrogen, Carlsbad, Calif.) in a total volume of 50 μl. Mutagenic PCRwas performed with 30 cycles of: 30 seconds of denaturing at 94° C.,followed by annealing at 55° C. for 1.5 min, and extension at 72° C. for4 min. The PCR product was digested by MseI and BamHI. This fragment wasligated with the 4.5-kb fragment of pQE-80L-Kana digested with MseI andBamHI. The resulting pQE-80L-Kana derivatives were used to transform E.coli BL21 (Novagen). Colonies were grown on a plate of LTB-agar medium(LB supplemented with 1% v/v tributyrin, 0.1% v/v Tween 80, 100 mg/L ofkanamycin, 0.01 μM isopropyl β-D-thiogalactopyranoside, and 1.5% agar)at 37° C.

Example 4: Expression of ActRIIBecd-Associated Polypeptides

Stably transformed E. coli cells as described in Example 3 were used toexpress ActRIIBecd-associated polypeptides (i.e., “domains”) from themutagenized DNA of Example 2.

A. Fermentation of Transformants

In one embodiment, overnight cultures (about 10 hrs) of E. coli BL21transformants with pQE-80L-Kana derivatives in 500 mL Erlenmeyer flaskscontaining 65 mL of medium (10 g/L BBL PhytonePeptone, 5 g/L BactoYeastExtract, 10 g/L NaCl) with 25-32 μg/mL of kanamycin were grown at 30° C.to 37° and agitated with 180±20 rpm. 37-420 mL of the overnight cultureswere added to 3.7-42 L of TB medium (BBL Phytone Peptone 18 g, Bactoyeast extract 36 g, KH₂PO₄ 18.81 g, Glycerol 6 mL in 1 L water)containing 23.8-38.5 μg/mL of kanamycin and 1-3 mmol/L isopropylβ-D-thiogalactopyranoside (IPTG) in 5-50 L fermentation tank andtemperature control ranging from 37° C. to 42° C., and the culture mediawas agitated with 260-450 rpm for 10-24 hours. The cells were collected,in an ice water bath, after centrifugation for 10 minutes at 8,000 rpmin a GSA rotor (Sorvall).

In another embodiment, 1 L of LB liquid medium (with 100 mg/L ofkanamycin) is inoculated with a freshly grown colony (E. coli BL21transformants with pQE-80L-Kana derivatives) or 10 mL of freshly grownculture and incubated at 37° C. until OD₆₀₀ reaches 0.4-0.8. Theexpression of the polypeptides is induced by adding 40 or 400 μM IPTGfor 3 to 5 hours at 37° C. After centrifugation (about 8,000 rpm), cellsare collected at 4° C.

B. Recovery and Purification of Polypeptides from E. coli

E. coli BL21/pQE-80L-Kana derivatives cells were fermented as previouslydescribed in Example 4A. In one embodiment, cell disruption and recoveryof polypeptides from those derivatives was performed at 4° C. About 18 gof wet cells were suspended in 60 mL of 0.1M TRIS/HCl, 10 mM EDTA(Ethylenediaminetetraacetic acid), 1 mM PMSF (Phenyl Methan SulphonylFluoride), pH 8.3 (disruption buffer). The cells were passed two timesthrough a Frenchpress (SLM Instruments, Inc.) according to themanufacturer's instructions and the volume was brought to 200 mL withthe disruption buffer. The suspension was centrifuged for 20 min at15,000 g. The pellet obtained was suspended in 100 mL disruption buffercontaining 1M NaCl and centrifuged for 10 min as above. The pellet wassuspended in 100 mL disruption buffer containing 1% Triton X-100(Pierce) and again centrifuged for 10 min as above. The washed pelletwas then suspended in 50 mL of 20 mM Tris/HCl, 1 mM EDTA, 1 mM PMSF, 1%DTT (Dithiothreitol) and homogenised in a Teflon tissue grinder. Theresulting suspension contained crude polypeptides in a non-soluble form.

10 mL of the polypeptide suspension obtained according to previousembodiment were acidified with 10% acetic acid to pH 2.5 and centrifugedin an Eppendorf centrifuge for 10 min at room temperature. Thesupernatant was chromatographed on a Sephacryl S-100 column (Pharmacia,2.6×78 cm) in 10% acetic acid at a flow rate of 1.4 mL/min. Fractionscontaining polypeptide eluting between appropriate time periods werepooled. This material was used for refolding to get biologically activepolypeptides or for further purification.

5 mg of polypeptide from previous embodiment was dissolved in 140 mL 50mM Tris/HCl pH 8.0, 1M NaCl, 5 mM EDTA, 2 mM reduced glutathione, 1 mMoxidised glutathione and 33 mM Chaps (Calbiochem). After 72 hours at 4°C., the pH of the solution was adjusted to pH 2.5 with HCl and themixture was concentrated 10 times by ultrafiltration on a YM 10 membrane(Amicon, Danvers, Mass., USA) in an Amicon stirred cell. Theconcentrated solution was diluted to the original volume with 10 mM HCland concentrated to a final volume of 10 mL by the same method. Theprecipitate formed was removed by centrifugation at 5000 g for 30minutes. The supernatant contained disulfide linked polypeptide asjudged by SDS-PAGE under non-reducing conditions. The biologicalactivity of the preparation was measured by BIAcore™ assay (Example 5).

The concentrated solution from the previous embodiment was applied at aflow rate of 1 mL/min onto a Mono S HR 5/5 column (Pharmacia)equilibrated in a mixture of 85% buffer A (20 mM sodium acetate, 30%isopropanol, pH 4.0) and 15% buffer B (buffer A containing 1M sodiumchloride). The column was then washed at the same flow rate keeping thebuffer mixture composition constant until the absorbance reading at 280nm has reached baseline level, followed by a linear gradient over 20minutes starting upon injection at the equilibration conditions andending with a mixture of 50% buffer A/50% buffer B. The biologicallyactive polypeptide was eluted about 9 minutes after the start of thegradient and collected. As judged by biological activity determination,and SDS-PAGE under non-reducing conditions.

In another embodiment, the polypeptides are prepared from inclusionbodies of collected cells in Example 4A. After extraction (50 mM sodiumacetate, pH 5, 8 M urea, 14 mM 2-mercaptoethanol) at room temperatureovernight and exhaustive dialysis against water, the polypeptides arerefolded, concentrated and enriched by Sephacryl S-100 HR column(Pharmacia) in 1% acetic acid or 5 mM HCl at a flow rate of 1.8 mL/min.It is finally purified by FPLC (Fractogel EMD SO₃ ⁻ 650, 50 mM sodiumacetate, pH 5, 30% 2-propanol) eluting with a NaCl gradient from 0 to1.5 M. Fractions containing polypeptide eluting between appropriate timeperiods are pooled. After dialysis against water, the polypeptides arefreeze/dried and stored at −20° C. The purity of the polypeptides isanalyzed by SDS-PAGE, followed by staining with Coomassie Brilliant BlueR.

In other embodiment, each 1 gram of cell pellet, derived for examplefrom the above Example 4A, was resuspended in 10-20 mL of 10 mMTRIS/HCl, 150 mM NaCl, 1 mM EDTA, 5 mM DTT, pH 8.0 (disruption buffer),and the cells burst by sonication, using a Misonix 54000 instrument,with a Enhance Booster #1 probe, at 30 A (instrument scale) for 5minutes. Optionally, the cell lysate mixture was clarified bycentrifugation (either 18,000×g for 20 min or 15,000×g for 30 min) andthe pellet was washed several times with 10-20 mL disruption buffercontaining 1 v/v % Triton X-100 and centrifuged for 10 min as above. Thecell lysate was dissolved with 100-200 mL disruption buffer containing 6M urea and centrifuged for 10 min as above and the supernatantcontaining the polypeptides was retained for further purification.

The previous mentioned supernatant was dissolved in refolding buffer(100 mL Tris/HCl pH 8.0, 500 mM Arginine-HCl, 5 mM EDTA, 25 mM Chaps, 2mM oxidised glutathione and 1 mM reduced glutathione). After 4-7 days atroom temperature, the polypeptides were purified by FPLC (Fractogel EMDSO₃ ⁻ 650, 20 mM sodium acetate, pH 4-5, 30% 2-propanol and 25 mM Chaps)eluting with a NaCl gradient from 0 to 3 M. Fractions containingpolypeptide eluting between appropriate time periods were pooled. Thepurity of the polypeptides was analyzed by SDS-PAGE, followed bystaining with Coomassie Brilliant Blue R.

In certain embodiments, the heterodimers of the present disclosure canbe prepared by co-expression in a transient expression system aspreviously mentioned in Example 3 and the heterodimers can be isolatedfrom the culture medium for screening in the assays of Example 5.

Example 5: In Vitro BIAcore™ Assay

Biosensor experiments. In one embodiment, experiments were performed ona BIAcore™ T100/T200 instrument (Pharmacia Biosensor AB) in themultichannel mode (serial flow path involving flow cells 1+2+3+4). Flowrate was 10 μl/min; temperature was 25° C.; and data were recorded at2.5 points/s. All four segments of a sensor chip CM5 were coated withstreptavidin (Sigma) to a density of 2000 pg/mm² (2000 resonance units)by the aminocoupling procedure. ActRIIBecd (10 mg) and amine-PEG3-Biotin(10 mg, Pierce, Rockford, Ill.) were dissolved in 200 μl H₂O and 10 mgNaCNBH₃ was added to prepare biotinylated ActRIIBecd. The reactionmixture was heated at 70° C. for 24 h, after that a further 10 mgNaCNBH₃ was added and the reaction was heated at 70° C. for another 24h. After cooling to room temperature, the mixture was desalted with thespin column (3,000 molecular weight cut off (MWCO)). BiotinylatedActRIIBecd was collected, freeze-dried and used for streptavidin (SA)chip preparation. Amino-biotinylated ActRIIBecd was then immobilizedindependently on flow cells 2-4 for 10 minutes at a flow rate of 5μL/min and a concentration of 20 μM in 10 mM sodium acetate, pH 4.0, ata density of 50-250 resonance units (RU). The stored polypeptides weredissolved in glycine buffer (2.5 g of glycine, 0.5 g of sucrose, 370 mgof L-glutamate, 10 mg of sodium chloride, and 10 mg of Tween 80 in 100mL water, pH 4.5) to prepare 10 mg/mL solution and then diluted withprevious glycine buffer to prepare analytes with various concentration.Sensograms were recorded during flow of the analyte (theActRIIBecd-associated polypeptides (i.e., domains) as previouslydescribed) first through flow cell 1 (control) then through flow cell 2(biotinylated ActRIIBecd). The sensogram obtained for flow cell 1 wassubtracted from the sensogram obtained for flow cell 2. The sensogramsobtained at 1.11, 3.33, 10, 30, and 90 nM analyte were evaluated forequilibrium binding, association rate, and dissociation rate by usingthe programs supplied with the instrument (BIA evaluation 2.1; SoftwareHandbook. 1995; Pharmacia Biosensor AB). Analytes and bovine serumalbumin (negative control) were listed in Table 2. Sequencing (primer5′-CTCGAGAAAT CATAAAAAAT TTATTTG-3′ (SEQ ID NO: 31)) of the pQE-80L-Kanaderivatives in the clone related to analyte, which has a higher affinityconstant than that of albumin, was performed with a Perkin-Elmer ABIAutomated DNA Sequencer as previously described.

TABLE 2 Clone Affinity constant No. DNA sequence Domain sequenceMean_([nM]) SD_([nM])  7 5′-GCTCAAGCCAAACA AQAKHKGYKRLKSNCKR NBCAAAGGGTATAAACGCC HPLY TTAAGTCCAATTGTAAA (SEQ ID NO: 33)AGGCACCCTTTGTAC-3′ (SEQ ID NO: 32)  8 5′-GGCCAAGCCAAACGGQAKRKGYKRLKSSCKR 45.12  ±15.39 CAAAGGGTATAAACGCC HPLY TTAAGTCCAGCTGTAAG(SEQ ID NO: 35) AGACACCCTTTGTAC-3′ (SEQ ID NO: 34)  9 5′-GCCCAAGCCAAACAAQAKHKGYKRLKSSCKR 52.41  ±16.71 TAAAGGGTATAAACGCC HPLY TTAAGTCCAGCTGTAAG(SEQ ID NO: 37) AGACACCCTTTGTAC-3′ (SEQ ID NO: 36) 10 5′-GCTCAAGCCAAACAAQAKHKQRKRLKSSCKR 36.39 ±12.12 CAAACAGCGGAAACGCC HPLY TTAAGTCCAGCTGTAAG(SEQ ID NO: 39) AGACACCCTTTGTAC-3′ (SEQ ID NO: 38) 11 5′-GCTCAAGCCAAACAAQAKHKGRKRLKSSCKR 63.72  ±23.17 CAAAGGTCGGAAACGCC HPLY TTAAGTCCAGCTGTAAG(SEQ ID NO: 41) AGACACCCTTTGTAC-3′ (SEQ ID NO: 40) 12 5′-GCTCAAGCCAAACAAQAKHKQYKRLKSSCKR 58.42 ±24.42 CAAACAGTACAAACGCC HPLY TTAAGTCCAGCTGTAAG(SEQ ID NO: 43) AGACACCCTTTGTAC-3′ (SEQ ID NO: 42) 13 5′-GTGGATTTCAAGGAVDFKDVGWNDHAVAPPG NB CGTTGGGTGGAATGACC YHAFYCHGECPHPLADHATGCTGTGGCACCGCCG LNSDNHAIVQTKVNSV GGGTATCACGCCTTCTA (SEQ ID NO: 45)TTGCCACGGAGAATGCC CGCATCCACTGGCTGAT CATCTGAACTCAGATAA CCATGCCATTGTTCAGACCAAGGTTAATTCTGTT- 3′ (SEQ ID NO: 44) 14 5′-GTGGATTTCAAGGAVDFKDVGWNDHAVAPPG 38.32  ±12.79 CGTTGGGTGGAATGACC YHAFYCHGECPFPLADHATGCTGTGGCACCGCCG LNSDNHAIVQTKVNSV GGGTATCACGCCTTCTA (SEQ ID NO: 47)TTGCCACGGAGAATGCC CGTTCCCACTGGCTGAT CATCTGAACTCAGATAA CCATGCCATTGTTCAGACCAAGGTTAATTCTGTT- 3′ (SEQ ID NO: 46) 15 5′-GTGGACTTCAGTGAVDFSDVGWNDWIVAPPG 39.45  ±14.11 CGTGGGGTGGAATGACT YHAFYCHGECPFPLADHGGATTGTGGCTCCCCCG LNSTNHAIVQTLVNSV GGGTATCACGCCTTTTA (SEQ ID NO: 49)CTGCCACGGAGAATGCC CTTTTCCTCTGGCTGAT CATCTGAACTCCACTAA TCATGCCATTGTTCAGACGTTGGTCAACTCTGTT- 3′ (SEQ ID NO: 48) 16 5′-GTGGATTTCAGCGAVDFSDVGWNDWAVAPPG 55.98 ±18.12 CGTTGGGTGGAATGACT YHAFYCHGECPFPLADHGGGCTGTGGCACCGCCG LNSDNHAIVQTLVNSV GGGTATCACGCCTTCTA (SEQ ID NO: 51)TTGCCACGGAGAATGCC CGTTCCCACTGGCTGAT CATCTGAACTCAGATAA CCATGCCATTGTTCAGACCCTCGTTAATTCTGTT- 3′ (SEQ ID NO: 50) 17 5′-GTGGATTTCAGCGAVDFSDVGWNDWIVAPPG 67.42 ±17.89 CGTTGGGTGGAATGACT YHAFYCHGECPFPLADHGGATCGTGGCACCGCCG LNSDNHAIVQTLVNSV GGGTATCACGCCTTCTA (SEQ ID NO: 53)TTGCCACGGAGAATGCC CGTTCCCACTGGCTGAT CATCTGAACTCAGATAA CCATGCCATTGTTCAGACCCTCGTTAATTCTGTT- 3′ (SEQ ID NO: 52) 18 5′-GTGGATTTCTCTGAVDFSDVGWNDHAVAPPG 64.12 ±16.78 CGTTGGGTGGAATGACC YHAFYCHGECPFPLADHATGCTGTGGCACCGCCG LNSTNHAIVQTLVNSV GGGTATCACGCCTTCTA (SEQ ID NO: 55)TTGCCACGGAGAATGCC CGTTCCCACTGGCTGAT CATCTGAACTCAACGAA CCATGCCATTGTTCAGACCCTTGTTAATTCTGTT- 3′ (SEQ ID NO: 54) 19 5′-AATAGCAAAGATCCNSKDPKACCVPTELSAP 40.12 ±13.69 CAAGGCATGCTGTGTCC SPLYLDENEKPVLKNYQCGACAGAACTCAGTGCC DMVVHGCGCR CCCAGCCCGCTGTACCT (SEQ ID NO: 57)TGACGAGAATGAGAAGC CTGTACTCAAGAACTAT CAGGACATGGTAGTCCA TGGGTGTGGGTGTCGC-3′ (SEQ ID NO: 56) 20 5′-AATAGCAAAATCCC NSKIPKACCQPTELSAP NBCAAGGCATGCTGTCAGC SPLYLDENEKPVLKNYQ CGACAGAACTCAGTGCC DMVVEGCGCRCCCAGCCCGCTGTACCT (SEQ ID NO: 59) TGACGAGAATGAGAAGC CTGTACTCAAGAACTATCAGGACATGGTAGTCGA AGGGTGTGGGTGTCGC- 3′ (SEQ ID NO: 58) 215′-AACTCTAAGATTCC NSKIPKACCVPTELSAI 37.12 ±11.88 TAAGGCATGCTGTGTCC SMLYLDENEKVVLKNYQ CGACAGAACTCAGTGCT DMVVEGCGCR ATCTCGATGCTGTACCT(SEQ ID NO: 61) TGACGAGAATGAAAAGG TTGTATTAAAGAACTAT CAGGACATGGTTGTGGAGGGTTGTGGGTGTCGC- 3′ (SEQ ID NO: 60) 22 5′-AATAGCAAAATCCCNSKIPKACCVPTELSAI 51.72 ±18.52 CAAGGCATGCTGTGTCC SPLYLDENEKVVLKNYQCGACAGAACTCAGTGCC DMVVHGCGCR ATAAGCCCGCTGTACCT (SEQ ID NO: 63)TGACGAGAATGAGAAGG TCGTACTCAAGAACTAT CAGGACATGGTAGTCCA TGGGTGTGGGTGTCGC-3′ (SEQ ID NO: 62) 23 5′-AATAGCAAAATACC NSKIPKACCVPTELSAI 64.41 ±17.42CAAGGCATGCTGTGTCC SMLYLDENEKPVLKNYQ CGACAGAACTCAGTGCC DMVVEGCGCRATTAGCATGCTGTACCT (SEQ ID NO: 65) TGACGAGAATGAGAAGC CTGTACTCAAGAACTATCAGGACATGGTAGTCGA AGGGTGTGGGTGTCGC- 3′ (SEQ ID NO: 64) 245′-AATAGCAAAGATCC NSKDPKACCVPTELSAI 56.74 ±13.59 CAAGGCATGCTGTGTCCSMLYLDENEKVVLKNYQ CGACAGAACTCAGTGCC DMVVHGCGCR ATAAGCATGCTGTACCT(SEQ ID NO: 67) TGACGAGAATGAGAAGG TGGTACTCAAGAACTAT CAGGACATGGTAGTCCATGGGTGTGGGTGTCGC- 3′ (SEQ ID NO: 66) 25 5′-ACGTATCCAGCCTCTYPASPKPMRHKMRSCA 41.09 ±14.15 TCCGAAGCCGATGAGGC CCAGGLRNRTGTVATAAAATGCGGAGCTGC (SEQ ID NO: 69) GCGTGCTGCGCCGGAGG TCTCCGGAACCGAACAGGGACAGTG-3′ (SEQ ID NO: 68) 26 5′-ACGTATCCCGCCTC TYPASPKPMRHSMRSCA 39.58±16.14 TCCGAAGCCGATGAGGC CCAGGLRNQTGTV ATTCAATGCGGAGCTGC (SEQ ID NO: 71)GCGTGCTGCGCCGGAGG TCTCCGGAACCAGACAG GGACAGTG-3′ (SEQ ID NO: 70) 275′-ACGTATCCAGCCTC TYPASPKPMRWKMRSCA 49.33  ±14.11 TCCGAAGCCGATGAGGTCCAGGLRNRTGTV GGAAGATGCGGAGCTGC (SEQ ID NO: 73) GCGTGCTGCGCCGGAGGTCTCCGGAACCGGACAG GGACAGTG-3′ (SEQ ID NO: 72) 28 5′-GAGCCCCTGGGCGGEPLGGARWEAFDVTDAV 53.78  ±14.42 CGCGCGCTGGGAAGCGT QSHRRSPRASRKCCLGLTCGACGTGACGGACGCG RAVTAS GTGCAGAGCCACCGCCG (SEQ ID NO: 75)CTCGCCACGAGCCTCCC GCAAGTGCTGCCTGGGG CTGCGCGCGGTGACGGC CTCG-3′(SEQ ID NO: 74) 29 5′-GAGCCCCTGGGCGG EPLGGARWEAFDVTDAV 57.89 ±13.44CGCGCGCTGGGAAGCGT QSHRRSQRASRKCCLVL TCGACGTGACGGACGCG RAVTASGTGCAGAGCCACCGCCG (SEQ ID NO: 77) CTCGCAGCGAGCCTCCC GCAAGTGCTGCCTGGTTCTGCGCGCGGTGACGGC CTCG-3′ (SEQ ID NO: 76) 30 5′-ACCGCCCTAGATGGTALDGTRGAQGSGGGGG 61.74 ±16.41 GACTCGGGGAGCGCAGG GGGGGGGGGGGGGGGAGGAAGCGGTGGTGGCGGC RGHGRRGRSRCSRKSLH GGTGGCGGTGGCGGCGG VDFKELGCGGCGGCGGCGGCGGCG (SEQ ID NO: 355) GCGGCGGCGGCGGCGCA GGCAGGGGCCACGGGCGCAGAGGCCGGAGCCGCT GCAGTCGCAAGTCACTG CACGTGGACTTTAAGGA GCTGGGC-3′(SEQ ID NO: 78) NB: Binding is below detection limit (KD > 1 mM)

Example 6: Production of Recombinant Polypeptides

To determine whether affinity constants could be enhanced, theindividual domains in Table 2 were fused with one another to producerecombinant polypeptides using the PCR-Fusion procedure described byAtanassov et al. (2009, Plant Methods, 5:14), with some modifications.PCR-fusion was carried out using Phusion DNA polymerase (Finnzymes;Finland) and a standard thermal cycler. Gateway recombination reactionswere performed with BP Clonase II and LR Clonase II enzyme mixes(Invitrogen). Competent E. coli DH5a cells, were prepared according toNojima et al. (1990, Gene, 96 (1): 23-28). Plasmid DNA and PCR fragmentswere purified using QIAprep® Spin Miniprep Kit and QIAquick® GelExtraction and PCR purification kits (Qiagen, Germany).

DNA template(s), PCR primers, and the DNA/polypeptide sequences of theresultant recombinant polypeptides are provided in Table 3. PCR-fusioninvolves two or three parallel PCR amplifications from plasmidtemplate(s). PCR fusion of the amplified fragments through a singleoverlap extension was carried out on gel purified PCR fragments fromthese parallel reactions. Cycling parameters were identical for all PCRamplifications in this manuscript using reaction mix and conditionsaccording to Phusion DNA polymerase guidelines (NewEnglandBiolabs:Phusion™ High-Fidelity DNA Polymerase. Manual). Annealing temperaturesfrom plasmid templates were 55° C.

For fusion of two PCR fragments, 30 μl overlap extension reactions wereused, which contained: 16 μl mixture of the two PCR fragments (normally8 μl for each one; approx. 200-800 ng, DNA), 6 μl of 5× Phusion HFBuffer, 3 μl of 2 mM dNTP mix, 0.3 μl of Phusion™ DNA Polymerase (2U/μl). No primers were added to the overlap extension mixture. Whenthree DNA fragments were fused, an 18 μl mixture of the PCR fragments(normally 6 μl for each one) was used. Generally, equal volumes ofpurified PCR fragments were used without checking exact DNAconcentrations. If the molar ratios of the amplified PCR fragmentsappeared to differ substantially (e.g., by more than 5-7 fold, followingestimation of DNA band intensities after agarose electrophoresis),volumes from purified PCR fragments were adjusted accordingly. Thereaction mix was incubated at 98° C. for 30 sec., 60° C. for 1 min and72° C. for 7 min. DNA obtained after the overlap extension reaction waspurified using a PCR purification kit. PCR products were digested andconed in the pQE-80L-Kana vector for protein/polypeptide expression aspreviously described. The affinity of purified protein/polypeptide toActRIIBecd was monitored by previously discussed BIAcore™ T100/T200 (GEHealthcare) and the data were analyzed by using BIAevaluation softwarever. 4.1 (GE Healthcare) in Example 5.

TABLE 3 Clone No. for DNA sequence/ Recombinant Affinity constantTemplate Polypeptide sequence Primers Mean[nm] SD[nm]  9 + 11 5′-First pair: 59.11 ±19.71 GCCCAAGCCAAACATAA 5′- AGGGTATAAACGCCTTATTAACCATGGCCCA AGTCCAGCTGTAAGAGA AGCCAAACAT-3′ CACCCTTTGTACGCTCAA(SEQ ID NO: 81) GCCAAACACAAAGGTCG 5′- GAAACGCCTTAAGTCCA GTTTGGCTTGAGCGTGCTGTAAGAGACACCCT ACAAAGGGTG-3′ TTGTAC-3′ (SEQ ID NO: (SEQ ID NO: 82)79)/ Second pair: AQAKHKGYKRLKSSCKR 5′- HPLYAQAKHKGRKRLKSCACCCTTTGTACGCT SCKRHPLY (SEQ ID NO: CAAGCCAAAC-3′ 80) (SEQ ID NO: 83)5′- GGATCCTTAGTACA AAGGGTGTCTC-3′ (SEQ ID NO: 84) 11 + 9 5′- First pair:58.22 ±18.15 GCTCAAGCCAAACACAA 5′- AGGTCGGAAACGCCTTA TTAACCATGGCTCAAAGTCCAGCTGTAAGAGA GCCAAACACA-3′ CACCCTTTGTACGCCCA (SEQ ID NO: 87)AGCCAAACATAAAGGGT 5′- ATAAACGCCTTAAGTCC GTTTGGCTTGGGCGTAGCTGTAAGAGACACCC ACAAAGGGTG-3′ TTTGTAC-3′ (SEQ ID NO: (SEQ ID NO: 88)85)/ Second pair: AQAKHKGRKRLKSSCKR 5′- HPLYAQAKHKGYKRLKSCACCCTTTGTACGCC SCKRHPLY (SEQ ID NO: CAAGCCAAAC-3′ 86) (SEQ ID NO: 89)5′- GGATCCTTAGTACA AAGGGTGTCTC-3′ (SEQ ID NO: 90) 17 + 18 5′-First pair: 65.77 ±19.56 GTGGATTTCAGCGACGT 5′- TGGGTGGAATGACTGGATTAACCATGGTGGAT TCGTGGCACCGCCGGGG TTCAGCGACG-3′ (SEQ TATCACGCCTTCTATTGCID NO: 93) CACGGAGAATGCCCGTT 5′- CCCACTGGCTGATCATCT CAGAGAAATCCACAGAACTCAGATAACCATG ACAGAATTAAC-3′ CCATTGTTCAGACCCTCG (SEQ ID NO: 94)TTAATTCTGTTGTGGATT Second pair: TCTCTGACGTTGGGTGG 5′- AATGACCATGCTGTGGCGTTAATTCTGTTGTG ACCGCCGGGGTATCACG GATTTCTCTG-3′ (SEQ CCTTCTATTGCCACGGAID NO:95) GAATGCCCGTTCCCACT 5′- GGCTGATCATCTGAACT GGATCCTTAAACAGCAACGAACCATGCCATT AATTAACAAGG-3′ GTTCAGACCCTTGTTAAT (SEQ ID NO: 96)TCTGTT-3′ (SEQ ID NO: 91)/ VDFSDVGWNDWIVAPPG YHAFYCHGECPFPLADHLNSDNHAIVQTLVNSVVDF SDVGWNDHAVAPPGYHA FYCHGECPFPLADHLNSTNHAIVQTLVNSV (SEQ ID NO: 92) 18 + 17 5′- First pair: 66.38 ±16.41GTGGATTTCTCTGACGTT 5′- GGGTGGAATGACCATGC TTAACCATGGTGGATTGTGGCACCGCCGGGGT TTCTCTGACG-3′ (SEQ ATCACGCCTTCTATTGCC ID NO: 99)ACGGAGAATGCCCGTTC 5′- CCACTGGCTGATCATCT CGCTGAAATCCACA GAACTCAACGAACCATGACAGAATTAAC-3′ CCATTGTTCAGACCCTTG (SEQ ID NO: 100) TTAATTCTGTTGTGGATTSecond pair: TCAGCGACGTTGGGTGG 5′- AATGACTGGATCGTGGC GTTAATTCTGTTGTGACCGCCGGGGTATCACG GATTTCAGCG-3′ CCTTCTATTGCCACGGA (SEQ ID NO: 101)GAATGCCCGTTCCCACT 5′- GGCTGATCATCTGAACT GGATCCTTAAACAG CAGATAACCATGCCATTAATTAACGAGG-3′ GTTCAGACCCTCGTTAAT (SEQ ID NO: 102) TCTGTT-3′ (SEQ ID NO:97)/ VDFSDVGWNDHAVAPPG YHAFYCHGECPFPLADHL NSTNHAIVQTLVNSVVDFSDVGWNDWIVAPPGYHA FYCHGECPFPLADHLNSD NHAIVQTLVNSV (SEQ ID NO: 98) 23 +24 5′- First pair: 61.77 ±15.74 AATAGCAAAATACCCAA 5′- GGCATGCTGTGTCCCGATTAACCATGAATAG CAGAACTCAGTGCCATT CAAAATACCCA-3′ AGCATGCTGTACCTTGA(SEQ ID NO: 105) CGAGAATGAGAAGCCTG 5′- TACTCAAGAACTATCAG GATCTTTGCTATTGCGACATGGTAGTCGAAGG GACACCCACAC-3′ GTGTGGGTGTCGCAATA (SEQ ID NO: 106)GCAAAGATCCCAAGGCA Second pair: TGCTGTGTCCCGACAGA 5′- ACTCAGTGCCATAAGCAGTGTGGGTGTCGCAA TGCTGTACCTTGACGAG TAGCAAAGATC-3′ AATGAGAAGGTGGTACT(SEQ ID NO: 107) CAAGAACTATCAGGACA 5′- TGGTAGTCCATGGGTGT GGATCCTTACGACAGGGTGTCGC-3′ (SEQ ID CCCACACCCAT-3′ NO: 103)/ (SEQ ID NO: 108)NSKIPKACCVPTELSAISM LYLDENEKPVLKNYQDM VVEGCGCRNSKDPKACCVPTELSAISMLYLDENEK VVLKNYQDMVVHGCGC R (SEQ ID NO: 104) 24 + 23 5′-First pair: 62.74 ±17.84 AATAGCAAAGATCCCAA 5′- GGCATGCTGTGTCCCGATTAACCATGAATAG CAGAACTCAGTGCCATA CAAAGATCCCA-3′ AGCATGCTGTACCTTGA(SEQ ID NO: 111) CGAGAATGAGAAGGTGG 5′- TACTCAAGAACTATCAG GTATTTTGCTATTGCGACATGGTAGTCCATGG GACACCCACAC-3′ GTGTGGGTGTCGCAATA (SEQ ID NO: 112)GCAAAATACCCAAGGCA Second pair: TGCTGTGTCCCGACAGA 5′- ACTCAGTGCCATTAGCAGTGTGGGTGTCGCAA TGCTGTACCTTGACGAG TAGCAAAATAC-3′ AATGAGAAGCCTGTACT(SEQ ID NO: 113) CAAGAACTATCAGGACA 5′- TGGTAGTCGAAGGGTGT GGATCCTTAGCGACGGGTGTCGC-3′ (SEQ ID ACCCACACCCT-3′ NO: 109)/ (SEQ ID NO: 114)NSKDPKACCVPTELSAIS MLYLDENEKVVLKNYQD MVVHGCGCRNSKIPKACCVPTELSAISMLYLDENE KPVLKNYQDMVVEGCGC R (SEQ ID NO: 110) 12 + 25 5′-First pair: 51.21 ±12.34 GCTCAAGCCAAACACAA 5′- ACAGTACAAACGCCTTATTAACCATGGCTCAA AGTCCAGCTGTAAGAGA GCCAAACACAA-3′ CACCCTTTGTACACGTAT(SEQ ID NO: 117) CCAGCCTCTCCGAAGCC 5′- GATGAGGCATAAAATGC GAGGCTGGATACGTGGAGCTGCGCGTGCTGC GTACAAAGGGTG-3′ GCCGGAGGTCTCCGGAA (SEQ ID NO: 118)CCGAACAGGGACAGTG- Second pair: 3′ (SEQ ID NO: 115)/ 5′-AQAKHKQYKRLKSSCKR CACCCTTTGTACACG HPLYTYPASPKPMRHKM TATCCAGCCTC-3′RSCACCAGGLRNRTGTV (SEQ ID NO:119) (SEQ ID NO: 116) 5′- GGATCCTTACACTGTCCCTGTTCGG-3′ (SEQ ID NO: 120) 25 + 12 5′- First pair: NBACGTATCCAGCCTCTCC 5′- GAAGCCGATGAGGCATA TTAACCATGACGTATAAATGCGGAGCTGCGCG CCAGCCTCTCC-3′ TGCTGCGCCGGAGGTCT (SEQ ID NO: 123)CCGGAACCGAACAGGGA 5′- CAGTGGCTCAAGCCAAA GTTTGGCTTGAGCCACACAAACAGTACAAACG CTGTCCCTGTTC-3′ CCTTAAGTCCAGCTGTA (SEQ ID NO: 124)AGAGACACCCTTTGTAC- Second pair: 3′ (SEQ ID NO: 121)/ 5′-TYPASPKPMRHKMRSCA GAACAGGGACAGTG CCAGGLRNRTGTVAQAK GCTCAAGCCAAAC-3′HKQYKRLKSSCKRHPLY (SEQ ID NO: 125) (SEQ ID NO: 122) 5′- GGATCCTTAGTACAAAGGGTGTCTC-3′ (SEQ ID NO: 126) 16 + 26 5′- First pair: 57.78 ±19.11GTGGATTTCAGCGACGT 5′- TGGGTGGAATGACTGGG TTAACCATGGTGGATCTGTGGCACCGCCGGGG TTCAGCGACG-3′ (SEQ TATCACGCCTTCTATTGC ID NO: 129)CACGGAGAATGCCCGTT 5′- CCCACTGGCTGATCATCT GGCGGGATACGTAAGAACTCAGATAACCATG CAGAATTAACG-3′ CCATTGTTCAGACCCTCG (SEQ ID NO: 130)TTAATTCTGTTACGTATC Second pair: CCGCCTCTCCGAAGCCG 5′- ATGAGGCATTCAATGCGCGTTAATTCTGTTAC GAGCTGCGCGTGCTGCG GTATCCCGCC-3′ (SEQ CCGGAGGTCTCCGGAACID NO: 131) CAGACAGGGACAGTG-3′ 5′- (SEQ ID NO: 127)/ GGATCCTTACACTGVDFSDVGWNDWAVAPP TCCCTGTCTGG-3′ GYHAFYCHGECPFPLADH (SEQ ID NO: 132)LNSDNHAIVQTLVNSVTY PASPKPMRHSMRSCACCA GGLRNQTGTV (SEQ ID NO: 128) 26 +16 5′- First pair: 61.74 ±13.69 ACGTATCCCGCCTCTCCG 5′- AAGCCGATGAGGCATTCTTAACCATGACGTAT AATGCGGAGCTGCGCGT CCCGCCTCTCC-3′ GCTGCGCCGGAGGTCTC(SEQ ID NO: 135) CGGAACCAGACAGGGAC 5′- AGTGGTGGATTTCAGCG CGCTGAAATCCACCAACGTTGGGTGGAATGAC CTGTCCCTGTC-3′ TGGGCTGTGGCACCGCC (SEQ ID NO: 136)GGGGTATCACGCCTTCT Second pair: ATTGCCACGGAGAATGC 5′- CCGTTCCCACTGGCTGATGACAGGGACAGTGG CATCTGAACTCAGATAA TGGATTTCAGCG-3′ CCATGCCATTGTTCAGA(SEQ ID NO: 137) CCCTCGTTAATTCTGTT- 5′- 3′ (SEQ ID NO: 133)/GGATCCTTAAACAG TYPASPKPMRHSMRSCAC AATTAACGAGGG-3′ CAGGLRNQTGTVVDFSD(SEQ ID NO: 138) VGWNDWAVAPPGYHAF YCHGECPFPLADHLNSDN HAIVQTLVNSV (SEQ IDNO: 134) 12 + 28 5′- First pair: 59.14 ±16.11 GCTCAAGCCAAACACAA 5′-ACAGTACAAACGCCTTA TTAACCATGGCTCAA AGTCCAGCTGTAAGAGA GCCAAACACAAAC-3′CACCCTTTGTACGAGCC (SEQ ID NO: 141) CCTGGGCGGCGCGCGCT 5′-GGGAAGCGTTCGACGTG CCGCCCAGGGGCTC ACGGACGCGGTGCAGAG GTACAAAGGGTGTC-CCACCGCCGCTCGCCAC 3′ (SEQ ID NO: 142) GAGCCTCCCGCAAGTGC Second pair:TGCCTGGGGCTGCGCGC 5′- GGTGACGGCCTCG-3′ GACACCCTTTGTACG (SEQ ID NO: 139)/AGCCCCTGGGCGG-3′ AQAKHKQYKRLKSSCKR (SEQ ID NO: 143) HPLYEPLGGARWEAFDV5′- TDAVQSHRRSPRASRKCC GGATCCTTACGAGG LGLRAVTAS (SEQ ID NO:CCGTCACCGCGCGC- 140) 3′ (SEQ ID NO: 144) 28 + 12 5′- First pair: 57.89±18.67 GAGCCCCTGGGCGGCGC 5′- GCGCTGGGAAGCGTTCG TTAACCATGGAGCCACGTGACGGACGCGGTG CCTGGGCGGCG-3′ CAGAGCCACCGCCGCTC (SEQ ID NO: 147)GCCACGAGCCTCCCGCA 5′- AGTGCTGCCTGGGGCTG GTTTGGCTTGAGCCGCGCGCGGTGACGGCCTC AGGCCGTCAC-3′ GGCTCAAGCCAAACACA (SEQ ID NO: 148)AACAGTACAAACGCCTT Second pair: AAGTCCAGCTGTAAGAG 5′- ACACCCTTTGTAC-3′GTGACGGCCTCGGCT (SEQ ID NO: 145)/ CAAGCCAAAC-3′ EPLGGARWEAFDVTDAV(SEQ ID NO: 149) QSHRRSPRASRKCCLGLR 5′- AVTASAQAKHKQYKRLK GGATCCTTAGTACASSCKRHPLY (SEQ ID NO: AAGGGTGTCTC-3′ 146) (SEQ ID NO: 150) 16 + 29 5′-First pair: 58.71 ±18.14 GTGGATTTCAGCGACGT 5′- TGGGTGGAATGACTGGGTTAACCATGGTGGAT CTGTGGCACCGCCGGGG TTCAGCGACG-3′ (SEQ TATCACGCCTTCTATTGCID NO: 153) CACGGAGAATGCCCGTT 5′- CCCACTGGCTGATCATCT GCCCAGGGGCTCAAGAACTCAGATAACCATG CAGAATTAACG-3′ CCATTGTTCAGACCCTCG (SEQ ID NO: 154)TTAATTCTGTTGAGCCCC Second pair: TGGGCGGCGCGCGCTGG 5′- GAAGCGTTCGACGTGACCGTTAATTCTGTTGA GGACGCGGTGCAGAGCC GCCCCTGGGC-3′ (SEQ ACCGCCGCTCGCAGCGAID NO: 155) GCCTCCCGCAAGTGCTG 5′- CCTGGTTCTGCGCGCGG GGATCCTTACGAGGTGACGGCCTCG-3′ (SEQ CCGTCACCGCG-3′ ID NO: 151)/ (SEQ ID NO: 156)VDFSDVGWNDWAVAPPG YHAFYCHGECPFPLADHL NSDNHAIVQTLVNSVEPLGGARWEAFDVTDAVQSH RRSQRASRKCCLVLRAVT AS (SEQ ID NO: 152) 29 + 16 5′-First pair: 54.31 ±12.89 GAGCCCCTGGGCGGCGC 5′- GCGCTGGGAAGCGTTCGTTAACCATGGAGCC ACGTGACGGACGCGGTG CCTGGGCGGCG-3′ CAGAGCCACCGCCGCTC(SEQ ID NO: 159) GCAGCGAGCCTCCCGCA 5′- AGTGCTGCCTGGTTCTGC GCTGAAATCCACCGGCGCGGTGACGGCCTCG AGGCCGTCACC-3′ GTGGATTTCAGCGACGT (SEQ ID NO: 160)TGGGTGGAATGACTGGG Second pair: CTGTGGCACCGCCGGGG 5′- TATCACGCCTTCTATTGCGGTGACGGCCTCGGT CACGGAGAATGCCCGTT GGATTTCAGC-3′ (SEQ CCCACTGGCTGATCATCTID NO: 161) GAACTCAGATAACCATG 5′- CCATTGTTCAGACCCTCG GGATCCTTAAACAGTTAATTCTGTT-3′ (SEQ ID AATTAACGAGG-3′ NO: 157)/ (SEQ ID NO: 162)EPLGGARWEAFDVTDAV QSHRRSQRASRKCCLVLR AVTASVDFSDVGWNDW AVAPPGYHAFYCHGECPFPLADHLNSDNHAIVQTLV NSV (SEQ ID NO: 158) 30 + 12 5′- First pair: 61.12±13.71 ACCGCCCTAGATGGGAC 5′- TCGGGGAGCGCAGGGAA TTAACCATGACCGCCGCGGTGGTGGCGGCGGT CTAGATGGGAC-3′ GGCGGTGGCGGCGGCGG (SEQ ID NO: 165)CGGCGGCGGCGGCGGCG 5′- GCGGCGGCGGCGCAGGC GTTTGGCTTGAGCGCAGGGGCCACGGGCGCAG CCAGCTCCTTA-3′ AGGCCGGAGCCGCTGCA (SEQ ID NO: 166)GTCGCAAGTCACTGCAC Second pair: GTGGACTTTAAGGAGCT 5′- GGGCGCTCAAGCCAAACTAAGGAGCTGGGCG ACAAACAGTACAAACGC CTCAAGCCAAAC-3′ CTTAAGTCCAGCTGTAA(SEQ ID NO: 167) GAGACACCCTTTGTAC-3′ 5′- (SEQ ID NO: 163)/GGATCCTTAGTACA TALDGTRGAQGSGGGGG AAGGGTGTCTCT-3′ GGGGGGGGGGGGGGGA(SEQ ID NO: 168) GRGHGRRGRSRCSRKSLH VDFKELGAQAKHKQYKRLKSSCKRHPLY (SEQ ID NO: 164) 12 + 30 5′- First pair: 59.11 ±12.47GCTCAAGCCAAACACAA 5′- ACAGTACAAACGCCTTA TTAACCATGGCTCAAAGTCCAGCTGTAAGAGA GCCAAACACA-3′ CACCCTTTGTACACCGCC (SEQ ID NO: 171)CTAGATGGGACTCGGGG 5′- AGCGCAGGGAAGCGGTG CATCTAGGGCGGTGTGTGGCGGCGGTGGCGGT ACAAAGGGTG-3′ GGCGGCGGCGGCGGCGG (SEQ ID NO: 172)CGGCGGCGGCGGCGGCG Second pair: GCGGCGCAGGCAGGGGC 5′- CACGGGCGCAGAGGCCGCACCCTTTGTACACC GAGCCGCTGCAGTCGCA GCCCTAGATG-3′ (SEQ AGTCACTGCACGTGGACID NO:173) TTTAAGGAGCTGGGC-3′ 5′- (SEQ ID NO: 169)/ GGATCCTTAGCCCAAQAKHKQYKRLKSSCKR GCTCCTTAAAG-3′ HPLYTALDGTRGAQGSG (SEQ ID NO: 174)GGGGGGGGGGGGGGGG GGGAGRGHGRRGRSRCS RKSLHVDFKELG (SEQ ID NO: 170) 16 + 305′- First pair: NB GTGGATTTCAGCGACGT 5′- TGGGTGGAATGACTGGGTTAACCATGGTGGAT CTGTGGCACCGCCGGGG TTCAGCGACG-3′ (SEQ TATCACGCCTTCTATTGCID NO: 177) CACGGAGAATGCCCGTT 5′- CCCACTGGCTGATCATCT CATCTAGGGCGGTAGAACTCAGATAACCATG ACAGAATTAAC-3′ CCATTGTTCAGACCCTCG (SEQ ID NO: 178)TTAATTCTGTTACCGCCC Second pair: TAGATGGGACTCGGGGA 5′- GCGCAGGGAAGCGGTGGGTTAATTCTGTTACC TGGCGGCGGTGGCGGTG GCCCTAGATG-3′ (SEQ GCGGCGGCGGCGGCGGCID NO: 179) GGCGGCGGCGGCGGCGG 5′- CGGCGCAGGCAGGGGCC GGATCCTTAGCCCAACGGGCGCAGAGGCCGG GCTCCTTAAAG-3′ AGCCGCTGCAGTCGCAA (SEQ ID NO: 180)GTCACTGCACGTGGACT TTAAGGAGCTGGGC-3′ (SEQ ID NO: 175)/ VDFSDVGWNDWAVAPPGYHAFYCHGECPFPLADHL NSDNHAIVQTLVNSVTAL DGTRGAQGSGGGGGGGG GGGGGGGGGGGGAGRGHGRRGRSRCSRKSLHVDF KELG (SEQ ID NO: 176) 12 + 16 5′- First pair: 67.42±19.51 GCTCAAGCCAAACACAA 5′- ACAGTACAAACGCCTTA TTAACCATGGCTCAAAGTCCAGCTGTAAGAGA GCCAAACACA-3′ CACCCTTTGTACGTGGAT (SEQ ID NO: 183)TTCAGCGACGTTGGGTG 5′- GAATGACTGGGCTGTGG CGCTGAAATCCACGTCACCGCCGGGGTATCAC ACAAAGGGTG-3′ GCCTTCTATTGCCACGG (SEQ ID NO: 184)AGAATGCCCGTTCCCAC Second pair: TGGCTGATCATCTGAAC 5′- TCAGATAACCATGCCATCACCCTTTGTACGTG TGTTCAGACCCTCGTTAA GATTTCAGCG-3′ (SEQ TTCTGTT-3′(SEQ ID NO: ID NO: 185) 181)/ 5′- AQAKHKQYKRLKSSCKR GGATCCTTAAACAGHPLYVDFSDVGWNDWA AATTAACGAGG-3′ VAPPGYHAFYCHGECPFP (SEQ ID NO: 186)LADHLNSDNHAIVQTLVN SV (SEQ ID NO: 182) 10 + 15 5′- First pair: 31.27±12.74 GCTCAAGCCAAACACAA 5′- ACAGCGGAAACGCCTTA TTAACCATGGCTCAAAGTCCAGCTGTAAGAGA GCCAAACACA-3′ CACCCTTTGTACGTGGA (SEQ ID NO: 189)CTTCAGTGACGTGGGGT 5′- GGAATGACTGGATTGTG CACTGAAGTCCACGTGCTCCCCCGGGGTATCA ACAAAGGGTG-3′ CGCCTTTTACTGCCACGG (SEQ ID NO: 190)AGAATGCCCTTTTCCTCT Second pair: GGCTGATCATCTGAACT 5′- CCACTAATCATGCCATTCACCCTTTGTACGTG GTTCAGACGTTGGTCAA GACTTCAGTG-3′ (SEQ CTCTGTT (SEQ ID NO:ID NO: 191) 187)/ 5′- AQAKHKQRKRLKSSCKR GGATCCTTAAACAG HPLYVDFSDVGWNDWIVAGTTGACCAAC-3′ APPGYHAFYCHGECPFPL (SEQ ID NO: 192) ADHLNSTNHAIVQTLVNSV (SEQ ID NO: 188) 15 + 10 5′- First pair: 29.74 ±13.51GTGGACTTCAGTGACGT 5′- GGGGTGGAATGACTGGA TTAACCATGGTGGA TTGTGGCTCCCCCGGGGCTTCAGTGACG-3′ TATCACGCCTTTTACTGC (SEQ ID NO: 195) CACGGAGAATGCCCTTT 5′-TCCTCTGGCTGATCATCT GTTTGGCTTGAGCAA GAACTCCACTAATCATG CAGAGTTGAC-3′CCATTGTTCAGACGTTG (SEQ ID NO: 196) GTCAACTCTGTTGCTCAA Second pair:GCCAAACACAAACAGCG 5′- GAAACGCCTTAAGTCCA GTCAACTCTGTTGCTGCTGTAAGAGACACCCT CAAGCCAAAC-3′ TTGTAC-3′ (SEQ ID NO: (SEQ ID NO: 197)193)/ 5′- VDFSDVGWNDWIVAPPG GGATCCTTAGTACA YHAFYCHGECPFPLADHLAAGGGTGTCTC-3′ NSTNHAIVQTLVNSVAQA (SEQ ID NO: 198) KHKQRKRLKSSCKRHPLY(SEQ ID NO: 194) 15 + 21 5′- First pair: 32.64 ±12.78 GTGGACTTCAGTGACGT5′- GGGGTGGAATGACTGGA TTAACCATGGTGGA TTGTGGCTCCCCCGGGG CTTCAGTGACG-3′TATCACGCCTTTTACTGC (SEQ ID NO: 201) CACGGAGAATGCCCTTT 5′-TCCTCTGGCTGATCATCT GAATCTTAGAGTTAA GAACTCCACTAATCATG CAGAGTTGAC-3′CCATTGTTCAGACGTTG (SEQ ID NO: 202) GTCAACTCTGTTAACTCT Second pair:AAGATTCCTAAGGCATG 5′- CTGTGTCCCGACAGAAC GTCAACTCTGTTAACTCAGTGCTATCTCGATGC TCTAAGATTC-3′ (SEQ TGTACCTTGACGAGAAT ID NO: 203)GAAAAGGTTGTATTAAA 5′- GAACTATCAGGACATGG GGATCCTTAGCGAC TTGTGGAGGGTTGTGGGACCCACAACCC-3′ TGTCGC (SEQ ID NO: 199)/ (SEQ ID NO: 204)VDFSDVGWNDWIVAPPG YHAFYCHGECPFPLADHL NSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYL DENEKVVLKNYQDMVVE GCGCR (SEQ ID NO: 200) 21 + 15 5′-First pair: 31.04 ±16.58 AACTCTAAGATTCCTAA 5′- GGCATGCTGTGTCCCGATTAACCATGAACTCT CAGAACTCAGTGCTATC AAGATTCCTA-3′ (SEQ TCGATGCTGTACCTTGACID NO: 207) GAGAATGAAAAGGTTGT 5′- ATTAAAGAACTATCAGG ACTGAAGTCCACGCACATGGTTGTGGAGGGT GACACCCACAA-3′ TGTGGGTGTCGCGTGGA (SEQ ID NO: 208)CTTCAGTGACGTGGGGT Second pair: GGAATGACTGGATTGTG 5′- GCTCCCCCGGGGTATCATTGTGGGTGTCGCGT CGCCTTTTACTGCCACGG GGACTTCAGT-3′ (SEQ AGAATGCCCTTTTCCTCTID NO: 209) GGCTGATCATCTGAACT 5′- CCACTAATCATGCCATT GGATCCTTAAACAGGTTCAGACGTTGGTCAA AGTTGACCAAC-3′ CTCTGTT-3′ (SEQ ID NO: (SEQ ID NO: 210)205)/ NSKIPKACCVPTELSAISM LYLDENEKVVLKNYQDM VVEGCGCRVDFSDVGWNDWIVAPPGYHAFYCHGE CPFPLADHLNSTNHAIVQ TLVNSV (SEQ ID NO: 206) 21 + 10 5′-First pair: 30.21 ±12.76 AACTCTAAGATTCCTAA 5′- GGCATGCTGTGTCCCGATTAACCATGAACTCT CAGAACTCAGTGCTATC AAGATTCCTA-3′ (SEQ TCGATGCTGTACCTTGACID NO: 213) GAGAATGAAAAGGTTGT 5′- ATTAAAGAACTATCAGG TTGGCTTGAGCGCGAACATGGTTGTGGAGGGT CACCCACAAC-3′ TGTGGGTGTCGCGCTCA (SEQ ID NO: 214)AGCCAAACACAAACAGC Second pair: GGAAACGCCTTAAGTCC 5′- AGCTGTAAGAGACACCCGTTGTGGGTGTCGCG TTTGTAC-3′ (SEQ ID NO: CTCAAGCCAA-3′ 211)/(SEQ ID NO: 215) NSKIPKACCVPTELSAISM 5′- LYLDENEKVVLKNYQDMGGATCCTTAGTACA VVEGCGCRAQAKHKQRK AAGGGTGTCTC-3′ RLKSSCKRHPLY (SEQ ID(SEQ ID NO: 216) NO: 212) 10 + 21 5′- First pair: 27.31 ±11.79GCTCAAGCCAAACACAA 5′- ACAGCGGAAACGCCTTA TTAACCATGGCTCAAAGTCCAGCTGTAAGAGA GCCAAACACA-3′ CACCCTTTGTACAACTCT (SEQ ID NO: 219)AAGATTCCTAAGGCATG 5′- CTGTGTCCCGACAGAAC GAATCTTAGAGTTGTTCAGTGCTATCTCGATGC ACAAAGGGTG-3′ TGTACCTTGACGAGAAT (SEQ ID NO: 220)GAAAAGGTTGTATTAAA Second pair: GAACTATCAGGACATGG 5′- TTGTGGAGGGTTGTGGGCACCCTTTGTACAAC TGTCGC (SEQ ID NO: 217)/ TCTAAGATTC-3′ (SEQAQAKHKQRKRLKSSCKR ID NO: 221) HPLYNSKIPKACCVPTELS 5′- AISMLYLDENEKVVLKNYGGATCCTTAGCGAC QDMVVEGCGCR (SEQ ID ACCCACAACCC-3′ NO: 218)(SEQ ID NO: 222)  8 + 14 5′- First pair: 35.14 ±13.64 GGCCAAGCCAAACGCAA5′- AGGGTATAAACGCCTTA TTAACCATGGGCCA AGTCCAGCTGTAAGAGA AGCCAAACGCA-3′CACCCTTTGTACGTGGAT (SEQ ID NO: 225) TTCAAGGACGTTGGGTG 5′-GAATGACCATGCTGTGG CCTTGAAATCCACGT CACCGCCGGGGTATCAC ACAAAGGGTG-3′GCCTTCTATTGCCACGG (SEQ ID NO: 226) AGAATGCCCGTTCCCAC Second pair:TGGCTGATCATCTGAAC 5′- TCAGATAACCATGCCAT CACCCTTTGTACGTGTGTTCAGACCAAGGTTA GATTTCAAGG-3′ (SEQ ATTCTGTT-3′ (SEQ ID NO: ID NO: 227)223)/ 5′- GQAKRKGYKRLKSSCKR GGATCCTTAAACAG HPLYVDFKDVGWNDHAVAATTAACCTTG-3′ APPGYHAFYCHGECPFPL (SEQ ID NO: 228) ADHLNSDNHAIVQTKVNSV (SEQ ID NO: 224) 14 + 8 5′- First pair: 33.79 ±16.51 GTGGATTTCAAGGACGT5′- TGGGTGGAATGACCATG TTAACCATGGTGGAT CTGTGGCACCGCCGGGG TTCAAGGACG-3′TATCACGCCTTCTATTGC (SEQ ID NO: 231) CACGGAGAATGCCCGTT 5′-CCCACTGGCTGATCATCT GTTTGGCTTGGCCAA GAACTCAGATAACCATG CAGAATTAAC-3′CCATTGTTCAGACCAAG (SEQ ID NO: 232) GTTAATTCTGTTGGCCAA Second pair:GCCAAACGCAAAGGGTA 5′- TAAACGCCTTAAGTCCA GTTAATTCTGTTGGCGCTGTAAGAGACACCCT CAAGCCAAAC-3′ TTGTAC-3′ (SEQ ID NO: (SEQ ID NO: 233)229)/ 5′- VDFKDVGWNDHAVAPPG GGATCCTTAGTACA YHAFYCHGECPFPLADHLAAGGGTGTCTC-3′ NSDNHAIVQTKVNSVGQ (SEQ ID NO: 234) AKRKGYKRLKSSCKRHPLY (SEQ ID NO: 230) 19 + 8 5′- First pair: 33.51 ±15.71 AATAGCAAAGATCCCAA5′- GGCATGCTGTGTCCCGA TTAACCATGAATAG CAGAACTCAGTGCCCCC CAAAGATCCCA-3′AGCCCGCTGTACCTTGA (SEQ ID NO: 237) CGAGAATGAGAAGCCTG 5′-TACTCAAGAACTATCAG TTGGCTTGGCCGCGA GACATGGTAGTCCATGG CACCCACACC-3′ (SEQGTGTGGGTGTCGCGGCC ID NO: 238) AAGCCAAACGCAAAGGG Second pair:TATAAACGCCTTAAGTC 5′- CAGCTGTAAGAGACACC GGTGTGGGTGTCGCG CTTTGTAC-3′(SEQ ID NO: GCCAAGCCAA-3′ 235)/ (SEQ ID NO: 239) NSKDPKACCVPTELSAPS 5′-PLYLDENEKPVLKNYQD GGATCCTTAGTACA MVVHGCGCRGQAKRKG AAGGGTGTCTC-3′YKRLKSSCKRHPLY (SEQ (SEQ ID NO: 240) ID NO: 236)  8 + 19 5′- First pair:31.12 ±13.42 GGCCAAGCCAAACGCAA 5′- AGGGTATAAACGCCTTA TTAACCATGGGCCAAGTCCAGCTGTAAGAGA AGCCAAACGCA-3′ CACCCTTTGTACAATAG (SEQ ID NO: 243)CAAAGATCCCAAGGCAT 5′- GCTGTGTCCCGACAGAA GATCTTTGCTATTGTCTCAGTGCCCCCAGCCC ACAAAGGGTG-3′ GCTGTACCTTGACGAGA (SEQ ID NO: 244)ATGAGAAGCCTGTACTC Second pair: AAGAACTATCAGGACAT 5′- GGTAGTCCATGGGTGTGCACCCTTTGTACAAT GGTGTCGC-3′ (SEQ ID AGCAAAGATC-3′ NO: 241)/(SEQ ID NO: 245) GQAKRKGYKRLKSSCKR 5′- HPLYNSKDPKACCVPTEL GGATCCTTAGCGACSAPSPLYLDENEKPVLKN ACCCACACCCA-3′ YQDMVVHGCGCR (SEQ (SEQ ID NO: 246)ID NO: 242) 19 + 14 5′- First pair: 32.09 ±13.03 AATAGCAAAGATCCCAA 5′-GGCATGCTGTGTCCCGA TTAACCATGAATAG CAGAACTCAGTGCCCCC CAAAGATCCCA-3′AGCCCGCTGTACCTTGA (SEQ ID NO: 249) CGAGAATGAGAAGCCTG 5′-TACTCAAGAACTATCAG CTTGAAATCCACGCG GACATGGTAGTCCATGG ACACCCACAC-3′GTGTGGGTGTCGCGTGG (SEQ ID NO: 250) ATTTCAAGGACGTTGGG Second pair:TGGAATGACCATGCTGT 5′- GGCACCGCCGGGGTATC GTGTGGGTGTCGCGTACGCCTTCTATTGCCACG GGATTTCAAG-3′ (SEQ GAGAATGCCCGTTCCCA ID NO: 251)CTGGCTGATCATCTGAA 5′- CTCAGATAACCATGCCA GGATCCTTAAACAG TTGTTCAGACCAAGGTTAATTAACCTTG-3′ AATTCTGTT-3′ (SEQ ID (SEQ ID NO: 252) NO: 247)/NSKDPKACCVPTELSAPS PLYLDENEKPVLKNYQD MVVHGCGCRVDFKDVG WNDHAVAPPGYHAFYCHGECPFPLADHLNSDNHAI VQTKVNSV (SEQ ID NO: 248) 14 + 19 5′- First pair:30.98 ±12.07 GTGGATTTCAAGGACGT 5′- TGGGTGGAATGACCATG TTAACCATGGTGGATCTGTGGCACCGCCGGGG TTCAAGGACG-3′ TATCACGCCTTCTATTGC (SEQ ID NO: 255)CACGGAGAATGCCCGTT 5′- CCCACTGGCTGATCATCT GATCTTTGCTATTAAGAACTCAGATAACCATG CAGAATTAAC-3′ CCATTGTTCAGACCAAG (SEQ ID NO: 256)GTTAATTCTGTTAATAGC Second pair: AAAGATCCCAAGGCATG 5′- CTGTGTCCCGACAGAACGTTAATTCTGTTAAT TCAGTGCCCCCAGCCCG AGCAAAGATC-3′ CTGTACCTTGACGAGAA(SEQ ID NO: 257) TGAGAAGCCTGTACTCA 5′- AGAACTATCAGGACATG GGATCCTTAGCGACGTAGTCCATGGGTGTGG ACCCACACCCA-3′ GTGTCGC-3′ (SEQ ID NO: (SEQ ID NO: 258)253)/ VDFKDVGWNDHAVAPPG YHAFYCHGECPFPLADHL NSDNHAIVQTKVNSVNSKDPKACCVPTELSAPSPLYL DENEKPVLKNYQDMVVH GCGCR (SEQ ID NO: 254) 10 + 15 +21 5′- First pair: 17.25 ±11.20 GCTCAAGCCAAACACAA 5′- ACAGCGGAAACGCCTTATTAACCATGGCTCAA AGTCCAGCTGTAAGAGA GCCAAACACA-3′ CACCCTTTGTACGTGGA(SEQ ID NO: 261) CTTCAGTGACGTGGGGT 5′- GGAATGACTGGATTGTG CACTGAAGTCCACGTGCTCCCCCGGGGTATCA ACAAAGGGTG-3′ CGCCTTTTACTGCCACGG (SEQ ID NO: 262)AGAATGCCCTTTTCCTCT Second pair: GGCTGATCATCTGAACT 5′- CCACTAATCATGCCATTCACCCTTTGTACGTG GTTCAGACGTTGGTCAA GACTTCAGTG-3′ (SEQ CTCTGTTAACTCTAAGATID NO: 263) TCCTAAGGCATGCTGTG 5′- TCCCGACAGAACTCAGT GAATCTTAGAGTTAAGCTATCTCGATGCTGTAC CAGAGTTGAC-3′ CTTGACGAGAATGAAAA (SEQ ID NO: 264)GGTTGTATTAAAGAACT Third pair: ATCAGGACATGGTTGTG 5′- GAGGGTTGTGGGTGTCGGTCAACTCTGTTAAC C-3′ (SEQ ID NO: 259)/ TCTAAGATTC-3′ (SEQAQAKHKQRKRLKSSCKR ID NO: 265) HPLYVDFSDVGWNDWIV 5′- APPGYHAFYCHGECPFPLGGATCCTTAGCGAC ADHLNSTNHAIVQTLVNS ACCCACAACCC-3′ VNSKIPKACCVPTELSAIS(SEQ ID NO: 266) MLYLDENEKVVLKNYQD MVVEGCGCR (SEQ ID NO: 260) 15 + 10 +21 5′- First pair: 15.14 ±13.21 GTGGACTTCAGTGACGT 5′- GGGGTGGAATGACTGGATTAACCATGGTGGA TTGTGGCTCCCCCGGGG CTTCAGTGACG-3′ TATCACGCCTTTTACTGC(SEQ ID NO: 269) CACGGAGAATGCCCTTT 5′- TCCTCTGGCTGATCATCTGTTTGGCTTGAGCAA GAACTCCACTAATCATG CAGAGTTGAC-3′ CCATTGTTCAGACGTTG(SEQ ID NO: 270) GTCAACTCTGTTGCTCAA Second pair: GCCAAACACAAACAGCG 5′-GAAACGCCTTAAGTCCA GTCAACTCTGTTGCT GCTGTAAGAGACACCCT CAAGCCAAAC-3′TTGTACAACTCTAAGATT (SEQ ID NO: 271) CCTAAGGCATGCTGTGT 5′-CCCGACAGAACTCAGTG GAATCTTAGAGTTGT CTATCTCGATGCTGTACC ACAAAGGGTG-3′TTGACGAGAATGAAAAG (SEQ ID NO: 272) GTTGTATTAAAGAACTA Third pair:TCAGGACATGGTTGTGG 5′- AGGGTTGTGGGTGTCGC- CACCCTTTGTACAAC 3′(SEQ ID NO: 267)/ TCTAAGATTC-3′ (SEQ VDFSDVGWNDWIVAPPG ID NO: 273)YHAFYCHGECPFPLADHL 5′- NSTNHAIVQTLVNSVAQA GGATCCTTAGCGACKHKQRKRLKSSCKRHPLY ACCCACAACCC-3′ NSKIPKACCVPTELSAISM (SEQ ID NO: 274)LYLDENEKVVLKNYQDM VVEGCGCR (SEQ ID NO: 268) 21 + 15 + 10 5′- First pair:14.21 ±13.51 AACTCTAAGATTCCTAA 5′- GGCATGCTGTGTCCCGA TTAACCATGAACTCTCAGAACTCAGTGCTATC AAGATTCCTA-3′ (SEQ TCGATGCTGTACCTTGAC ID NO: 277)GAGAATGAAAAGGTTGT 5′- ATTAAAGAACTATCAGG CACTGAAGTCCACGCACATGGTTGTGGAGGGT GACACCCACA-3′ TGTGGGTGTCGCGTGGA (SEQ ID NO: 278)CTTCAGTGACGTGGGGT Second pair: GGAATGACTGGATTGTG 5′- GCTCCCCCGGGGTATCATGTGGGTGTCGCGTG CGCCTTTTACTGCCACGG GACTTCAGTG-3′ (SEQ AGAATGCCCTTTTCCTCTID NO: 279) GGCTGATCATCTGAACT 5′- CCACTAATCATGCCATT GTTTGGCTTGAGCAAGTTCAGACGTTGGTCAA CAGAGTTGAC-3′ CTCTGTTGCTCAAGCCA (SEQ ID NO: 280)AACACAAACAGCGGAAA Third pair: CGCCTTAAGTCCAGCTG 5′- TAAGAGACACCCTTTGTGTCAACTCTGTTGCT AC-3′ (SEQ ID NO: 275)/ CAAGCCAAAC-3′NSKIPKACCVPTELSAISM (SEQ ID NO: 281) LYLDENEKVVLKNYQDM 5′-VVEGCGCRVDFSDVGWN GGATCCTTAGTACA DWIVAPPGYHAFYCHGE AAGGGTGTCTC-3′CPFPLADHLNSTNHAIVQ (SEQ ID NO: 282) TLVNSVAQAKHKQRKRLKSSCKRHPLY (SEQ ID NO: 276) 21 + 10 + 15 5′- First pair: 16.71 ±14.25AACTCTAAGATTCCTAA 5′- GGCATGCTGTGTCCCGA TTAACCATGAACTCTCAGAACTCAGTGCTATC AAGATTCCTA-3′ (SEQ TCGATGCTGTACCTTGAC ID NO: 285)GAGAATGAAAAGGTTGT 5′- ATTAAAGAACTATCAGG GTTTGGCTTGAGCGCACATGGTTGTGGAGGGT GACACCCACA-3′ TGTGGGTGTCGCGCTCA (SEQ ID NO: 286)AGCCAAACACAAACAGC Second pair: GGAAACGCCTTAAGTCC 5′- AGCTGTAAGAGACACCCTGTGGGTGTCGCGCT TTTGTACGTGGACTTCAG CAAGCCAAAC-3′ TGACGTGGGGTGGAATG(SEQ ID NO: 287) ACTGGATTGTGGCTCCC 5′- CCGGGGTATCACGCCTT CACTGAAGTCCACGTTTACTGCCACGGAGAAT ACAAAGGGTG-3′ GCCCTTTTCCTCTGGCTG (SEQ ID NO: 288)ATCATCTGAACTCCACT Third pair: AATCATGCCATTGTTCA 5′- GACGTTGGTCAACTCTGCACCCTTTGTACGTG TT-3′ (SEQ ID NO: 283)/ GACTTCAGTG-3′ (SEQNSKIPKACCVPTELSAISM ID NO: 289) LYLDENEKVVLKNYQDM 5′- VVEGCGCRAQAKHKQRKGGATCCTTAAACAG RLKSSCKRHPLYVDFSDV AGTTGACCAAC-3′ GWNDWIVAPPGYHAFYC(SEQ ID NO: 290) HGECPFPLADHLNSTNHA IVQTLVNSV (SEQ ID NO: 284)  8 + 14 +19 5′- First pair: 15.64 ±13.24 GGCCAAGCCAAACGCAA 5′- AGGGTATAAACGCCTTATTAACCATGGGCCA AGTCCAGCTGTAAGAGA AGCCAAACGCA-3′ CACCCTTTGTACGTGGAT(SEQ ID NO: 293) TTCAAGGACGTTGGGTG 5′- GAATGACCATGCTGTGG CCTTGAAATCCACGTCACCGCCGGGGTATCAC ACAAAGGGTG-3′ GCCTTCTATTGCCACGG (SEQ ID NO: 294)AGAATGCCCGTTCCCAC Second pair: TGGCTGATCATCTGAAC 5′- TCAGATAACCATGCCATCACCCTTTGTACGTG TGTTCAGACCAAGGTTA GATTTCAAGG-3′ (SEQ ATTCTGTTAATAGCAAAID NO: 295) GATCCCAAGGCATGCTG 5′- TGTCCCGACAGAACTCA GATCTTTGCTATTAAGTGCCCCCAGCCCGCTG CAGAATTAAC-3′ TACCTTGACGAGAATGA (SEQ ID NO: 296)GAAGCCTGTACTCAAGA Third pair: ACTATCAGGACATGGTA 5′- GTCCATGGGTGTGGGTGGTTAATTCTGTTAAT TCGC-3′ (SEQ ID NO: 291)/ AGCAAAGATC-3′GQAKRKGYKRLKSSCKR (SEQ ID NO: 297) HPLYVDFKDVGWNDHAV 5′-APPGYHAFYCHGECPFPL GGATCCTTAGCGAC ADHLNSDNHAIVQTKVNS ACCCACACCCA-3′VNSKDPKACCVPTELSAP (SEQ ID NO: 298) SPLYLDENEKPVLKNYQDMVVHGCGCR (SEQ ID NO: 292) 14 + 8 + 19 5′- First pair: 17.65 ±14.78GTGGATTTCAAGGACGT 5′- TGGGTGGAATGACCATG TTAACCATGGTGGATCTGTGGCACCGCCGGGG TTCAAGGACG-3′ TATCACGCCTTCTATTGC (SEQ ID NO: 301)CACGGAGAATGCCCGTT 5′- CCCACTGGCTGATCATCT GTTTGGCTTGGCCAAGAACTCAGATAACCATG CAGAATTAAC-3′ CCATTGTTCAGACCAAG (SEQ ID NO: 302)GTTAATTCTGTTGGCCAA Second pair: GCCAAACGCAAAGGGTA 5′- TAAACGCCTTAAGTCCAGTTAATTCTGTTGGC GCTGTAAGAGACACCCT CAAGCCAAAC-3′ TTGTACAATAGCAAAGA(SEQ ID NO: 303) TCCCAAGGCATGCTGTG 5′- TCCCGACAGAACTCAGT GATCTTTGCTATTGTGCCCCCAGCCCGCTGTA ACAAAGGGTG-3′ CCTTGACGAGAATGAGA (SEQ ID NO: 304)AGCCTGTACTCAAGAAC Third pair: TATCAGGACATGGTAGT 5′- CCATGGGTGTGGGTGTCCACCCTTTGTACAAT GC-3′ (SEQ ID NO: 299)/ AGCAAAGATC-3′ VDFKDVGWNDHAVAPPG(SEQ ID NO: 305) YHAFYCHGECPFPLADHL 5′- NSDNHAIVQTKVNSVGQ GGATCCTTAGCGACAKRKGYKRLKSSCKRHPL ACCCACACCCA-3′ YNSKDPKACCVPTELSAP (SEQ ID NO: 306)SPLYLDENEKPVLKNYQD MVVHGCGCR (SEQ ID NO: 300) 19 + 8 + 14 5′-First pair: 15.97 ±12.01 AATAGCAAAGATCCCAA 5′- GGCATGCTGTGTCCCGATTAACCATGAATAG CAGAACTCAGTGCCCCC CAAAGATCCCA-3′ AGCCCGCTGTACCTTGA(SEQ ID NO: 309) CGAGAATGAGAAGCCTG 5′- TACTCAAGAACTATCAG GTTTGGCTTGGCCGCGACATGGTAGTCCATGG GACACCCACA-3′ GTGTGGGTGTCGCGGCC (SEQ ID NO: 310)AAGCCAAACGCAAAGGG Second pair: TATAAACGCCTTAAGTC 5′- CAGCTGTAAGAGACACCTGTGGGTGTCGCGGC CTTTGTACGTGGATTTCA CAAGCCAAAC-3′ AGGACGTTGGGTGGAAT(SEQ ID NO: 311) GACCATGCTGTGGCACC 5′- GCCGGGGTATCACGCCT CCTTGAAATCCACGTTCTATTGCCACGGAGAA ACAAAGGGTG-3′ TGCCCGTTCCCACTGGCT (SEQ ID NO: 312)GATCATCTGAACTCAGA Third pair: TAACCATGCCATTGTTCA 5′- GACCAAGGTTAATTCTGCACCCTTTGTACGTG TT-3′ (SEQ ID NO: 307)/ GATTTCAAGG-3′ (SEQNSKDPKACCVPTELSAPS ID NO: 313) PLYLDENEKPVLKNYQD 5′- MVVHGCGCRGQAKRKGGGATCCTTAAACAG YKRLKSSCKRHPLYVDFK AATTAACCTTG-3′ DVGWNDHAVAPPGYHAF(SEQ ID NO: 314) YCHGECPFPLADHLNSDN HAIVQTKVNSV (SEQ ID NO: 308) 19 +14 + 8 5′- First pair: 14.12 ±10.27 AATAGCAAAGATCCCAA 5′-GGCATGCTGTGTCCCGA TTAACCATGAATAG CAGAACTCAGTGCCCCC CAAAGATCCCA-3′AGCCCGCTGTACCTTGA (SEQ ID NO: 317) CGAGAATGAGAAGCCTG 5′-TACTCAAGAACTATCAG CTTGAAATCCACGCG GACATGGTAGTCCATGG ACACCCACAC-3′GTGTGGGTGTCGCGTGG (SEQ ID NO: 318) ATTTCAAGGACGTTGGG Second pair:TGGAATGACCATGCTGT 5′- GGCACCGCCGGGGTATC GTGTGGGTGTCGCGTACGCCTTCTATTGCCACG GGATTTCAAG-3′ (SEQ GAGAATGCCCGTTCCCA ID NO: 319)CTGGCTGATCATCTGAA 5′- CTCAGATAACCATGCCA GTTTGGCTTGGCCAATTGTTCAGACCAAGGTT CAGAATTAAC-3′ AATTCTGTTGGCCAAGC (SEQ ID NO: 320)CAAACGCAAAGGGTATA Third pair: AACGCCTTAAGTCCAGC 5′- TGTAAGAGACACCCTTTGTTAATTCTGTTGGC GTAC-3′ (SEQ ID NO: 315)/ CAAGCCAAAC-3′NSKDPKACCVPTELSAPS (SEQ ID NO: 321) PLYLDENEKPVLKNYQD 5′-MVVHGCGCRVDFKDVG GGATCCTTAGTACA WNDHAVAPPGYHAFYCH AAGGGTGTCTC-3′GECPFPLADHLNSDNHAI (SEQ ID NO: 322) VQTKVNSVGQAKRKGYKRLKSSCKRHPLY (SEQ ID NO: 316) 10 + 14 + 21 5′- First pair: 17.98 ±11.61GCTCAAGCCAAACACAA 5′- ACAGCGGAAACGCCTTA TTAACCATGGCTCAAAGTCCAGCTGTAAGAGA GCCAAACAC-3′ (SEQ CACCCTTTGTACGTGGAT ID NO: 325)TTCAAGGACGTTGGGTG 5′- GAATGACCATGCTGTGG CTTGAAATCCACGTACACCGCCGGGGTATCAC CAAAGGGTG-3′ (SEQ GCCTTCTATTGCCACGG ID NO: 326)AGAATGCCCGTTCCCAC Second pair: TGGCTGATCATCTGAAC 5′- TCAGATAACCATGCCATCACCCTTTGTACGTG TGTTCAGACCAAGGTTA GATTTCAAG-3′ (SEQ ATTCTGTTAACTCTAAGAID NO: 327) TTCCTAAGGCATGCTGT 5′- GTCCCGACAGAACTCAG GAATCTTAGAGTTAATGCTATCTCGATGCTGTA CAGAATTAAG-3′ CCTTGACGAGAATGAAA (SEQ ID NO: 328)AGGTTGTATTAAAGAAC Third pair: TATCAGGACATGGTTGT 5′- GGAGGGTTGTGGGTGTCGTTAATTCTGTTAAC GC-3′ (SEQ ID NO: 323)/ TCTAAGATTC-3′ (SEQAQAKHKQRKRLKSSCKR ID NO: 329) HPLYVDFKDVGWNDHAV 5′- APPGYHAFYCHGECPFPLGGATCCTTAGCGAC ADHLNSDNHAIVQTKVNS ACCCACAACCC-3′ VNSKIPKACCVPTELSAIS(SEQ ID NO: 330) MLYLDENEKVVLKNYQD MVVEGCGCR (SEQ ID NO: 324)  8 + 15 +19 5′- First pair: 16.49 ±12.04 GGCCAAGCCAAACGCAA 5′- AGGGTATAAACGCCTTATTAACCATGGGCCA AGTCCAGCTGTAAGAGA AGCCAAACGC-3′ CACCCTTTGTACGTGGA(SEQ ID NO: 333) CTTCAGTGACGTGGGGT 5′- GGAATGACTGGATTGTG ACTGAAGTCCACGTAGCTCCCCCGGGGTATCA CAAAGGGTC-3′ (SEQ CGCCTTTTACTGCCACGG ID NO: 334)AGAATGCCCTTTTCCTCT Second pair: GGCTGATCATCTGAACT 5′- CCACTAATCATGCCATTCACCCTTTGTACGTG GTTCAGACGTTGGTCAA GACTTCAGT-3′ (SEQ CTCTGTTAATAGCAAAGID NO: 335) ATCCCAAGGCATGCTGT 5′- GTCCCGACAGAACTCAG GATCTTTGCTATTAATGCCCCCAGCCCGCTGT CAGAGTTGAC-3′ ACCTTGACGAGAATGAG (SEQ ID NO: 336)AAGCCTGTACTCAAGAA Third pair: CTATCAGGACATGGTAG 5′- TCCATGGGTGTGGGTGTGTCAACTCTGTTAAT CGC-3′ (SEQ ID NO: 331)/ AGCAAAGATC-3′ GQAKRKGYKRLKSSCKR(SEQ ID NO: 337) HPLYVDFSDVGWNDWIV 5′- APPGYHAFYCHGECPFPL GGATCCTTAGCGACADHLNSTNHAIVQTLVNS ACCCACACCCA-3′ VNSKDPKACCVPTELSAP (SEQ ID NO: 338)SPLYLDENEKPVLKNYQD MVVHGCGCR (SEQ ID NO: 332) 10 + 19 + 14 5′-First pair: 16.98 ±13.97 GCTCAAGCCAAACACAA 5′- ACAGCGGAAACGCCTTATTAACCATGGCTCAA AGTCCAGCTGTAAGAGA GCCAAACACA-3′ CACCCTTTGTACAATAG(SEQ ID NO: 341) CAAAGATCCCAAGGCAT 5′- GCTGTGTCCCGACAGAA GATCTTTGCTATTGTCTCAGTGCCCCCAGCCC ACAAAGGGTG-3′ GCTGTACCTTGACGAGA (SEQ ID NO: 342)ATGAGAAGCCTGTACTC Second pair: AAGAACTATCAGGACAT 5′- GGTAGTCCATGGGTGTGCACCCTTTGTACAAT GGTGTCGCGTGGATTTC AGCAAAGATC-3′ AAGGACGTTGGGTGGAA(SEQ ID NO: 343) TGACCATGCTGTGGCAC 5′- CGCCGGGGTATCACGCC CTTGAAATCCACGCGTTCTATTGCCACGGAGA ACACCCACAC-3′ ATGCCCGTTCCCACTGG (SEQ ID NO: 344)CTGATCATCTGAACTCA Third pair: GATAACCATGCCATTGT 5′- TCAGACCAAGGTTAATTGTGTGGGTGTCGCGT CTGTT-3′ (SEQ ID NO: 339)/ GGATTTCAAG-3′ (SEQAQAKHKQRKRLKSSCKR ID NO: 345) HPLYNSKDPKACCVPTEL 5′- SAPSPLYLDENEKPVLKNGGATCCTTAAACAG YQDMVVHGCGCRVDFK AATTAACCTTG-3′ DVGWNDHAVAPPGYHAF(SEQ ID NO: 346) YCHGECPFPLADHLNSDN HAIVQTKVNSV (SEQ ID NO: 340)  8 +21 + 15 5′- First pair: 17.11 ±12.10 GGCCAAGCCAAACGCAA 5′-AGGGTATAAACGCCTTA TTAACCATGGGCCA AGTCCAGCTGTAAGAGA AGCCAAACGC-3′CACCCTTTGTACAACTCT (SEQ ID NO: 349) AAGATTCCTAAGGCATG 5′-CTGTGTCCCGACAGAAC GAATCTTAGAGTTGT TCAGTGCTATCTCGATGC ACAAAGGGTG-3′TGTACCTTGACGAGAAT (SEQ ID NO: 350) GAAAAGGTTGTATTAAA Second pair:GAACTATCAGGACATGG 5′- TTGTGGAGGGTTGTGGG CACCCTTTGTACAACTGTCGCGTGGACTTCAG TCTAAGATTC-3′ (SEQ TGACGTGGGGTGGAATG ID NO: 351)ACTGGATTGTGGCTCCC 5′- CCGGGGTATCACGCCTT TCACTGAAGTCCACGTTACTGCCACGGAGAAT CGACACCCAC-3′ GCCCTTTTCCTCTGGCTG (SEQ ID NO: 352)ATCATCTGAACTCCACT Third pair: AATCATGCCATTGTTCA 5′- GACGTTGGTCAACTCTGGTGGGTGTCGCGTGG TT-3′ (SEQ ID NO: 347)/ ACTTCAGTGA-3′ (SEQGQAKRKGYKRLKSSCKR ID NO: 353) HPLYNSKIPKACCVPTELS 5′- AISMLYLDENEKVVLKNYGGATCCTTAAACAG QDMVVEGCGCRVDFSDV AGTTGACCAAC-3′ GWNDWIVAPPGYHAFYC(SEQ ID NO: 354) HGECPFPLADHLNSTNHA IVQTLVNSV (SEQ ID NO: 348) NB:Binding is below detection limit (KD > 1 mM)

The data shows that the affinity constant (K_(D)) was lower forrecombinant polypeptides formed from the following combinations of twoclones as compared to the individual polypeptides from each singleclone: clone no. 10 operably linked with clone no. 15 (SEQ ID NO: 188),clone no. 15 operably linked with clone no. 10 (SEQ ID NO: 194), cloneno. 15 operably linked with clone no. 21 (SEQ ID NO: 200), clone no. 21operably linked with clone no. 15 (SEQ ID NO: 206), clone no. 21operably linked with clone no. 10 (SEQ ID NO: 212), clone no. 10operably linked with clone no. 21 (SEQ ID NO: 218), clone no. 8 operablylinked with clone no. 14 (SEQ ID NO: 224), clone no. 14 operably linkedwith clone no. 8 (SEQ ID NO: 230), clone no. 19 operably linked withclone no. 8 (SEQ ID NO: 236), clone no. 8 operably linked with clone no.19 (SEQ ID NO: 242), clone no. 19 operably linked with clone no. 14 (SEQID NO: 248), and clone no. 14 operably linked with clone no. 19 (SEQ IDNO: 254). In other words, the recombinant polypeptides resulting fromthe noted combinations had a higher affinity against ActRIIBecd than theindividual polypeptides from each of clone no. 8 (SEQ ID NO: 35), cloneno. 10 (SEQ ID NO: 39), clone no. 14 (SEQ ID NO: 47), clone no. 15 (SEQID NO: 49), clone no. 19 (SEQ ID NO: 57), and clone no. 21 (SEQ ID NO:61).

In addition, recombinant polypeptides were produced from combinations ofthree clones using clone no. 8 (SEQ ID NO: 35), clone no. 10 (SEQ ID NO:39), clone no. 14 (SEQ ID NO: 47), clone no. 15 (SEQ ID NO: 49), cloneno. 19 (SEQ ID NO: 57), and clone no. 21 (SEQ ID NO: 61). Surprisingly,the K_(D) of recombinant polypeptides formed from the followingcombinations of three clones was lower than for polypeptides from theindividual clones or from combinations of two clones: clone no. 10operably linked with clone nos. 15 and 21 (SEQ ID NO: 260), clone no. 15operably linked with clone nos. 10 and 21 (SEQ ID NO: 268), clone no. 21operably linked with clone nos. 15 and 10 (SEQ ID NO: 276), clone no. 21operably linked with clone nos. 10 and 15 (SEQ ID NO: 284), clone no. 8operably linked with clone nos. 14 and 19 (SEQ ID NO: 292), clone no. 14operably linked with clone nos. 8 and 19 (SEQ ID NO: 300), clone no. 19operably linked with clone nos. 8 and 14 (SEQ ID NO: 308), clone no. 19operably linked with clone nos. 14 and 8 (SEQ ID NO: 316), clone no. 10operably linked with clone nos. 14 and 21 (SEQ ID NO: 324), clone no. 8operably linked with clone nos. 15 and 19 (SEQ ID NO: 332), clone no. 10operably linked with clone nos. 19 and 14 (SEQ ID NO: 340), and cloneno. 8 operably linked with clone nos. 21 and 15 (SEQ ID NO: 348).

Example 7: Post-Translation Modification

The effect of post-translation modification (PTM) on K_(D) values of therecombinant polypeptides was investigated. One example of PTM isdisulfide bond connection. Data showing the relation between disulfidebond location and binding affinity is provided in TABLE 4, which showsthat PTM affects the binding affinity of the recombinant polypeptidesagainst ActRIIBecd. The PTM assay was performed according to thefollowing experiments.

A. Enzymatic Digestion and Dimethyl Labeling

Polypeptides were prepared as in Examples 4 and 6. Standard proteinswere purchased from Sigma (St. Louise, Mo.). Optionally, 5 mM NEM(N-ethylmaleimide) (Sigma) in 100 mM sodium acetate (J. T. Baker,Phillipsburg, N.J.), pH 6, was used to block free cysteines at roomtemperature for 30 min. Enzymatic digestion was performed directly insodium acetate at 37° C. overnight with 1:50 trypsin (Promega, Madison,Wis.). Protein digest was diluted three times with 100 mM sodium acetate(pH 5) before dimethyl labeling.

In certain embodiments, recombinant polypeptides prepared as in Examples4 and 6 were diluted with 50 mM TEABC (Triethylammonium bicarbonate,T7408, Sigma-Aldrich) buffer (pH7) and split into two tubes fordifferent enzymatic digestion. First, NEM (N-ethylmaleimide, E3876,Sigma-Aldrich) was added at a final concentration of 5 mM to block freecysteines. The alkylation reaction was performed for 30 min at roomtemperature. After NEM alkylation, one tube was added with trypsin(V5111, Promega) (1:65) at 37° C. for 18 hrs followed by Glu-C (P8100S,New England BioLabs) (1:50) digestion at 37° C. overnight. Another onewas added with Glu-C(1:50) at 37° C. for 18 hrs followed by chymotrypsin(1:50) digestion at 37° C. overnight.

To perform dimethyl labeling, 2.5 μL of 4% (w/v) formaldehyde-H₂ (J. T.Baker) or 2.5 μL of 4% (w/v) formaldehyde-D₂ (Aldrich) was added to 504of protein digest followed by the addition of 2.5 μL of 600 mM sodiumcyanoborohydride (Sigma), and the reaction was performed at pH 5-6 for30 min.

B. Mass Spectrometry

ESI Q-TOF equipped with a CapLC system (Waters, Milford, Mass.)utilizing a capillary column (75 μm i.d., 10 cm in length, Csun, Taiwan)was used to perform the survey scan (MS, m/z 400-1600; MS/MS, m/z50-2000). The alkylated and dimethyl-labeled protein digest was subjectto LC-MS/MS analysis with a linear gradient from 5% to 50% acetonitrilecontaining 0.1% formic acid over 45 min.

In certain embodiments, the digested and dimethyl labeled protein digestwere analyzed with Q-Exactive Plus mass spectrometer coupled withUltimate 3000 RSLC system. The LC separation was performed using the C18column (Acclaim PepMap RSLC, 75 μm×150 mm, 2 μm, 100 Å) with thegradient shown below:

Time Flow (min) A % B % (μL/min) 0 99 1 0.25 6 99 1 0.25 45 70 30 0.2548 40 60 0.25 50 20 80 0.25 60 20 80 0.25 65 99 1 0.25 70 99 1 0.25

Mobile phase A: 0.1% formic acid

Mobile phase B: 95% acetonitrile/0.1% formic acid

Full MS scan was performed with the range of m/z 300-2000, and the tenmost intense ions from MS scan were subjected to fragmentation for MS/MSspectra.

C. Data Analysis.

MassLynx 4.0 was used to produce peak lists from raw data (subtract 30%,smooth 3/2 Savitzky Golay and center three channels 80% centroid). Arelatively high subtraction can be applied to eliminate backgroundnoise. True a₁ ions usually appear as major peaks so that they can bekept in the peak list.

D. Reversed-Phase Chromatography.

An Agilent 1100 HPLC system with a binary pump was equipped with a UVdetector and an autosampler. The proteins were injected onto a Zorbax300SB C8 column (150_2.1 mm, 5_m, 300 A) operated at 75° C. The flowrate was 0.5 ml/min. Mobile-phase A was water containing 0.1%trifluoroacetic acid. Mobile-phase B was 70% isopropyl alcohol, 20%acetonitrile, and aqueous 0.1% trifluoroacetic acid. Samples wereinjected at a loading condition of 10% B and increased to 19% B over 2min. Alinear elution gradient of 1.1% B/min started at 2 min and endedat 24 min. The column was then flushed for 5 min with 95% B. The columnwas reequilibrated with the loading condition for 5 min. This method wasable to partially resolve disulfide isoforms.

TABLE 4 Recombinant Polypeptide Identified Cysteine Affinity ConstantSEQ ID NO Sequence Pairs Mean[nm] SD[nm] 188 AQAKHKQRKRLKSSCKRHC15-C44^(a) 42.32 ±2.12 PLYVDFSDVGWNDWIVAP PGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSV 188 AQAKHKQRKRLKSSCKRH C44-C48^(a) 21.47 ±1.72PLYVDFSDVGWNDWIVAP PGYHAFYCHGECPFPLADH LNSTNHAIVQTLVNSV 194VDFSDVGWNDWIVAPPGY C23-C65^(a) 45.98 ±2.01 HAFYCHGECPFPLADHLNSTNHAIVQTLVNSVAQAKH KQRKRLKSSCKRHPLY 194 VDFSDVGWNDWIVAPPGY C23-C27^(a)18.14 ±1.67 HAFYCHGECPFPLADHLNS TNHAIVQTLVNSVAQAKH KQRKRLKSSCKRHPLY 200VDFSDVGWNDWIVAPPGY C23-C27^(a), C58-C91^(a), 18.62 ±1.41HAFYCHGECPFPLADHLNS C59-C93^(a) TNHAIVQTLVNSVNSKIPK ACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCG CR 200 VDFSDVGWNDWIVAPPGY C23-C27^(a), C58-C93^(a),20.13 ±2.06 HAFYCHGECPFPLADHLNS C59-C91^(a) TNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDEN EKVVLKNYQDMVVEGCG CR 200 VDFSDVGWNDWIVAPPGYC23-C27^(a), C58-C91^(b), 19.75 ±1.98 HAFYCHGECPFPLADHLNS C59-C93^(b)TNHAIVQTLVNSVNSKIPK ACCVPTELSAISMLYLDEN EKVVLKNYQDMVVEGCG CR 200VDFSDVGWNDWIVAPPGY C23-C58^(a) 47.62 ±2.89 HAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPK ACCVPTELSAISMLYLDEN EKVVLKNYQDMVVEGCG CR 206NSKIPKACCVPTELSAISML C67-C71^(a), C8-C41^(a), 15.49 ±3.12YLDENEKVVLKNYQDMV C9-C43^(a) VEGCGCRVDFSDVGWND WIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVN SV 206 NSKIPKACCVPTELSAISML C8-C71^(a) 49.66 ±1.09YLDENEKVVLKNYQDMV VEGCGCRVDFSDVGWND WIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVN SV 206 NSKIPKACCVPTELSAISML C67-C71^(a), C8-C43^(a),17.73 ±1.70 YLDENEKVVLKNYQDMV C9-C41^(a) VEGCGCRVDFSDVGWNDWIVAPPGYHAFYCHGECPF PLADHLNSTNHAIVQTLVN SV 206 NSKIPKACCVPTELSAISMLC67-C71^(a), C8-C43^(b), 16.97 ±2.15 YLDENEKVVLKNYQDMV C9-C41^(b)VEGCGCRVDFSDVGWND WIVAPPGYHAFYCHGECPF PLADHLNSTNHAIVQTLVN SV 212NSKIPKACCVPTELSAISML C59-C59^(b), C8-C41^(a), 18.13 ±2.21YLDENEKVVLKNYQDMV C9-C43^(a) VEGCGCRAQAKHKQRKRL KSSCKRHPLY 212NSKIPKACCVPTELSAISML C59-C59^(b), C8-C43^(a), 19.34 ±1.56YLDENEKVVLKNYQDMV C9-C41^(a) VEGCGCRAQAKHKQRKRL KSSCKRHPLY 212NSKIPKACCVPTELSAISML C41-C59^(a) 44.33 ±3.09 YLDENEKVVLKNYQDMVVEGCGCRAQAKHKQRKRL KSSCKRHPLY 218 AQAKHKQRKRLKSSCKRHC15-C15^(b), C29-C62^(a), 16.22 ±1.62 PLYNSKIPKACCVPTELSAI C30-C64^(a)SMLYLDENEKVVLKNYQD MVVEGCGCR 218 AQAKHKQRKRLKSSCKRHC15-C15^(b), C29-C64^(a), 18.42 ±1.79 PLYNSKIPKACCVPTELSAI C30-C62^(a)SMLYLDENEKVVLKNYQD MVVEGCGCR 218 AQAKHKQRKRLKSSCKRH C15-C30^(a) 40.89±2.62 PLYNSKIPKACCVPTELSAI SMLYLDENEKVVLKNYQD MVVEGCGCR 224GQAKRKGYKRLKSSCKRH C44-C48^(a) 22.31 ±1.99 PLYVDFKDVGWNDHAVAPPGYHAFYCHGECPFPLADH LNSDNHAIVQTKVNSV 224 GQAKRKGYKRLKSSCKRH C15-C44^(a)48.93 ±2.88 PLYVDFKDVGWNDHAVAP PGYHAFYCHGECPFPLADH LNSDNHAIVQTKVNSV 230VDFKDVGWNDHAVAPPG C23-C27^(a) 17.94 ±2.31 YHAFYCHGECPFPLADHLNSDNHAIVQTKVNSVGQAK RKGYKRLKSSCKRHPLY 230 VDFKDVGWNDHAVAPPG C27-C65^(a)52.23 ±1.63 YHAFYCHGECPFPLADHLN SDNHAIVQTKVNSVGQAK RKGYKRLKSSCKRHPLY 236NSKDPKACCVPTELSAPSP C59-C59^(b), C8-C41^(a), 16.21 ±2.10LYLDENEKPVLKNYQDMV C9-C43^(a) VHGCGCRGQAKRKGYKRL KSSCKRHPLY 236NSKDPKACCVPTELSAPSP C41-C59^(a) 49.51 ±3.30 LYLDENEKPVLKNYQDMVVHGCGCRGQAKRKGYKRL KSSCKRHPLY 236 NSKDPKACCVPTELSAPSPC59-C59^(b), C8-C43^(a), 17.05 ±1.27 LYLDENEKPVLKNYQDMV C9-C41^(a)VHGCGCRGQAKRKGYKRL KSSCKRHPLY 242 GQAKRKGYKRLKSSCKRHC15-C15^(b), C29-C62^(a), 17.78 ?’. 09 PLYNSKDPKACCVPTELSA C30-C64^(a)PSPLYLDENEKPVLKNYQD MVVHGCGCR 242 GQAKRKGYKRLKSSCKRH C29-C30^(a) 48.66±3.15 PLYNSKDPKACCVPTELSA PSPLYLDENEKPVLKNYQD MVVHGCGCR 242GQAKRKGYKRLKSSCKRH C15-C15^(b), C29-C64^(a), 18.11 ±1.63PLYNSKDPKACCVPTELSA C30-C62^(a) PSPLYLDENEKPVLKNYQD MVVHGCGCR 248NSKDPKACCVPTELSAPSP C67-C71^(a), C8-C41^(a), 18.52 ±1.74LYLDENEKPVLKNYQDMV C9-C43a VHGCGCRVDFKDVGWND HAVAPPGYHAFYCHGECPFPLADHLNSDNHAIVQTKVN SV 248 NSKDPKACCVPTELSAPSP C41-C43^(a) 47.81 ±3.22LYLDENEKPVLKNYQDMV VHGCGCRVDFKDVGWND HAVAPPGYHAFYCHGECPFPLADHLNSDNHAIVQTKVN SV 248 NSKDPKACCVPTELSAPSP C67-C71^(a), C8-C43^(a),19.98 ±2.14 LYLDENEKPVLKNYQDMV C9-C41^(a) VHGCGCRVDFKDVGWNDHAVAPPGYHAFYCHGECPF PLADHLNSDNHAIVQTKVN SV 248 NSKDPKACCVPTELSAPSPC67-C71^(a), C8-C43^(b), 19.25 ?'. 01 LYLDENEKPVLKNYQDMV C9-C41^(b)VHGCGCRVDFKDVGWND HAVAPPGYHAFYCHGECPF PLADHLNSDNHAIVQTKVN SV 254VDFKDVGWNDHAVAPPG C23-C27^(a), C58-C91^(a), 17.55 ±1.74YHAFYCHGECPFPLADHLN C59-C93^(a) SDNHAIVQTKVNSVNSKDP KACCVPTELSAPSPLYLDENEKPVLKNYQDMVVHGC GCR 254 VDFKDVGWNDHAVAPPG C23-C27^(a), C58-C93^(a),19.06 ±2.39 YHAFYCHGECPFPLADHLN C59-C91^(a) SDNHAIVQTKVNSVNSKDPKACCVPTELSAPSPLYLDE NEKPVLKNYQDMVVHGC GCR 254 VDFKDVGWNDHAVAPPGC23-C58^(a) 44.43 ±2.05 YHAFYCHGECPFPLADHLN SDNHAIVQTKVNSVNSKDPKACCVPTELSAPSPLYLDE NEKPVLKNYQDMVVHGC GCR 254 VDFKDVGWNDHAVAPPGC23-C27^(a), C58-C91^(b), 18.73 ±1.65 YHAFYCHGECPFPLADHLN C59-C93^(b)SDNHAIVQTKVNSVNSKDP KACCVPTELSAPSPLYLDE NEKPVLKNYQDMVVHGC GCR 260AQAKHKQRKRLKSSCKRH C15-C80, C44- 6.56 ±1.12 PLYVDFSDVGWNDWIVAPC112^(a), C48-C114^(a), PGYHAFYCHGECPFPLADH C79-C79^(b)LNSTNHAIVQTLVNSVNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 260AQAKHKQRKRLKSSCKRH C79-C80^(a) 29.14 ±1.35 PLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADH LNSTNHAIVQTLVNSVNSK IPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGC GCR 260 AQAKHKQRKRLKSSCKRH C15-C15^(b), C79- 7.64±1.03 PLYVDFSDVGWNDWIVAP C114^(a), C80-C112^(a), PGYHAFYCHGECPFPLADHC44-C48^(a) LNSTNHAIVQTLVNSVNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGCGCR 260 AQAKHKQRKRLKSSCKRH C80-C114^(a), C79- 8.31 ±1.07PLYVDFSDVGWNDWIVAP C112^(a), C44-C48^(a) PGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 260AQAKHKQRKRLKSSCKRH C15-C15^(b), C80- 7.45 ±1.67 PLYVDFSDVGWNDWIVAPC114^(a), C79-C112^(a), PGYHAFYCHGECPFPLADH C44-C48^(a)LNSTNHAIVQTLVNSVNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 260AQAKHKQRKRLKSSCKRH C80-C114^(b), C79- 6.89 ±0.96 PLYVDFSDVGWNDWIVAPC112^(b), C44-C48^(a) PGYHAFYCHGECPFPLADH LNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 268 VDFSDVGWNDWIVAPPGYC23-C27^(a), C65-C65^(b), 7.21 ±1.32 HAFYCHGECPFPLADHLNSC79-C112^(a), C80- TNHAIVQTLVNSVAQAKH C114^(a) KQRKRLKSSCKRHPLYNSKIPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 268 VDFSDVGWNDWIVAPPGYC23-C79^(a) 29.57 ±2.71 HAFYCHGECPFPLADHLNS TNHAIVQTLVNSVAQAKHKQRKRLKSSCKRHPLYNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 268VDFSDVGWNDWIVAPPGY C23-C27^(a), C65-C65^(b), 8.74 ±2.08HAFYCHGECPFPLADHLNS C79-C114^(a), C80- TNHAIVQTLVNSVAQAKH C112^(a)KQRKRLKSSCKRHPLYNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 268VDFSDVGWNDWIVAPPGY C23-C27^(a), C65-C65^(b), 7.96 ±0.75HAFYCHGECPFPLADHLNS C79-C112^(b), C80- TNHAIVQTLVNSVAQAKH C114^(b)KQRKRLKSSCKRHPLYNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 268VDFSDVGWNDWIVAPPGY C23-C27^(a), C65-C65^(b), 8.03 ±2.45HAFYCHGECPFPLADHLNS C79-C114^(b), C80- TNHAIVQTLVNSVAQAKH C112^(b)KQRKRLKSSCKRHPLYNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 276NSKIPKACCVPTELSAISML C67-C71^(a), C8-C41^(a), 5.71 ±1.60YLDENEKVVLKNYQDMV C9-C43^(a), C109-C109^(b) VEGCGCRVDFSDVGWNDWIVAPPGYHAFYCHGECPF PLADHLNSTNHAIVQTLVN SVAQAKHKQRKRLKSSCK RHPLY 276NSKIPKACCVPTELSAISML C8-C9^(a) 27.93 ±2.35 YLDENEKVVLKNYQDMVVEGCGCRVDFSDVGWND WIVAPPGYHAFYCHGECPF PLADHLNSTNHAIVQTLVNSVAQAKHKQRKRLKSSCK RHPLY 276 NSKIPKACCVPTELSAISMLC67-C71^(a), C8-C43^(a), 7.66 ±1.05 YLDENEKVVLKNYQDMVC9-C41^(a), C109-C109^(b) VEGCGCRVDFSDVGWND WIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVN SVAQAKHKQRKRLKSSCK RHPLY 276 NSKIPKACCVPTELSAISMLC67-C71^(a), C8-C43^(b), 6.31 ±1.38 YLDENEKVVLKNYQDMVC9-C41^(b), C109-C109^(b) VEGCGCRVDFSDVGWND WIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVN SVAQAKHKQRKRLKSSCK RHPLY 276 NSKIPKACCVPTELSAISMLC67-C71^(a), C8-C41^(b), 8.13 ±1.77 YLDENEKVVLKNYQDMVC9-C43^(b), C109-C109^(b) VEGCGCRVDFSDVGWND WIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVN SVAQAKHKQRKRLKSSCK RHPLY 284 NSKIPKACCVPTELSAISMLC59-C59^(b), C8-C41^(a), 4.89 ±1.13 YLDENEKVVLKNYQDMVC9-C43^(a), C88-C92^(a) VEGCGCRAQAKHKQRKRL KSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGE CPFPLADHLNSTNHAIVQT LVNSV 284 NSKIPKACCVPTELSAISMLC8-C9^(a) 31.79 ±3.15 YLDENEKVVLKNYQDMV VEGCGCRAQAKHKQRKRLKSSCKRHPLYVDFSDVGW NDWIVAPPGYHAFYCHGE CPFPLADHLNSTNHAIVQT LVNSV 284NSKIPKACCVPTELSAISML C59-C59^(b), C8-C43^(a), 6.88 ±1.35YLDENEKVVLKNYQDMV C9-C41^(a), C88-C92^(a) VEGCGCRAQAKHKQRKRLKSSCKRHPLYVDFSDVGW NDWIVAPPGYHAFYCHGE CPFPLADHLNSTNHAIVQT LVNSV 284NSKIPKACCVPTELSAISML C59-C59^(b), C8-C41^(b), 5.79 ±1.76YLDENEKVVLKNYQDMV C9-C43^(b), C88-C92^(a) VEGCGCRAQAKHKQRKRLKSSCKRHPLYVDFSDVGW NDWIVAPPGYHAFYCHGE CPFPLADHLNSTNHAIVQT LVNSV 284NSKIPKACCVPTELSAISML C59-C59^(b), C8-C43^(b), 6.91 ±1.55YLDENEKVVLKNYQDMV C9-C41^(b), C88-C92^(a) VEGCGCRAQAKHKQRKRLKSSCKRHPLYVDFSDVGW NDWIVAPPGYHAFYCHGE CPFPLADHLNSTNHAIVQT LVNSV 292GQAKRKGYKRLKSSCKRH C15-C80^(a), C44- 4.77 ±0.67 PLYVDFKDVGWNDHAVAPC112^(a), C48-C114^(a), PGYHAFYCHGECPFPLADH C79-C79^(b)LNSDNHAIVQTKVNSVNSK DPKACCVPTELSAPSPLYL DENEKPVLKNYQDMVVHG CGCR 292GQAKRKGYKRLKSSCKRH C79-C80^(a) 28.99 ±2.66 PLYVDFKDVGWNDHAVAPPGYHAFYCHGECPFPLADH LNSDNHAIVQTKVNSVNSK DPKACCVPTELSAPSPLYLDENEKPVLKNYQDMVVHG CGCR 292 GQAKRKGYKRLKSSCKRH C15-C15^(b), C79- 5.81±3.57 PLYVDFKDVGWNDHAVAP C114^(a), C80-C112^(a), PGYHAFYCHGECPFPLADHC44-C48^(a) LNSDNHAIVQTKVNSVNSK DPKACCVPTELSAPSPLYL DENEKPVLKNYQDMVVHGCGCR 292 GQAKRKGYKRLKSSCKRH C80-C114^(a), C79- 6.19 ±1.37PLYVDFKDVGWNDHAVAP C112^(a), C44-C48^(a) PGYHAFYCHGECPFPLADHLNSDNHAIVQTKVNSVNSK DPKACCVPTELSAPSPLYL DENEKPVLKNYQDMVVHG CGCR 292GQAKRKGYKRLKSSCKRH C80-C114^(b), C79- 5.54 ±1.30 PLYVDFKDVGWNDHAVAPC112^(b), C44-C48^(a) PGYHAFYCHGECPFPLADH LNSDNHAIVQTKVNSVNSKDPKACCVPTELSAPPLYL DENEKPVLKNYQDMVVHG CGCR 300 VDFKDVGWNDHAVAPPGC23-C27^(a), C65-C65^(b), 5.14 ±1.39 YHAFYCHGECPFPLADHLNC79-C112^(a), C80- SDNHAIVQTKVNSVGQAK C114^(a) RKGYKRLKSSCKRHPLYNSKDPKACCVPTELSAPSPLY LDENEKPVLKNYQDMVVH GCGCR 300 VDFKDVGWNDHAVAPPGC23-C79^(a) 32.69 ±2.45 YHAFYCHGECPFPLADHLN SDNHAIVQTKVNSVGQAKRKGYKRLKSSCKRHPLYNS KDPKACCVPTELSAPSPLY LDENEKPVLKNYQDMVVH GCGCR 300VDFKDVGWNDHAVAPPG C23-C27^(a), C65-C65^(b), 7.37 ±1.71YHAFYCHGECPFPLADHLN C79-C114^(a), C80- SDNHAIVQTKVNSVGQAK C112^(a)RKGYKRLKSSCKRHPLYNS KDPKACCVPTELSAPSPLY LDENEKPVLKNYQDMVVH GCGCR 300VDFKDVGWNDHAVAPPG C23-C27^(a), C65-C65^(b), 6.44 ±1.72YHAFYCHGECPFPLADHLN C79-C112^(b), C80- SDNHAIVQTKVNSVGQAK C114^(b)RKGYKRLKSSCKRHPLYNS KDPKACCVPTELSAPSPLY LDENEKPVLKNYQDMVVH GCGCR 308NSKDPKACCVPTELSAPSP C59-C59^(b), C8-C41^(a), 5.98 ±1.43LYLDENEKPVLKNYQDMV C9-C43^(a), C88-C92^(a) VHGCGCRGQAKRKGYKRLKSSCKRHPLYVDFKDVGW NDHAVAPPGYHAFYCHGE CPFPLADHLNSDNHAIVQT KVNSV 308NSKDPKACCVPTELSAPSP C8-C9^(a) 31.22 ±2.07 LYLDENEKPVLKNYQDMVVHGCGCRGQAKRKGYKRL KSSCKRHPLYVDFKDVGW NDHAVAPPGYHAFYCHGECPFPLADHLNSDNHAIVQT KVNSV 308 NSKDPKACCVPTELSAPSPC59-C59^(b), C8-C43^(a), 6.11 ±1.33 LYLDENEKPVLKNYQDMVC9-C41^(a), C88-C92^(a) VHGCGCRGQAKRKGYKRL KSSCKRHPLYVDFKDVGWNDHAVAPPGYHAFYCHGE CPFPLADHLNSDNHAIVQT KVNSV 308 NSKDPKACCVPTELSAPSPC59-C59^(b), C8-C41^(b), 6.73 ±1.55 LYLDENEKPVLKNYQDMVC9-C43^(b), C88-C92^(a) VHGCGCRGQAKRKGYKRL KSSCKRHPLYVDFKDVGWNDHAVAPPGYHAFYCHGE CPFPLADHLNSDNHAIVQT KVNSV 316 NSKDPKACCVPTELSAPSPC67-C71^(a), C8-C41^(a), 5.34 ±1.37 LYLDENEKPVLKNYQDMVC9-C43^(a), C109-C109^(b) VHGCGCRVDFKDVGWND HAVAPPGYHAFYCHGECPFPLADHLNSDNHAIVQTKVN SVGQAKRKGYKRLKSSCK RHPLY 316 NSKDPKACCVPTELSAPSPC8-C67^(a) 28.91 ±2.65 LYLDENEKPVLKNYQDMV VHGCGCRVDFKDVGWNDHAVAPPGYHAFYCHGECPF PLADHLNSDNHAIVQTKVN SVGQAKRKGYKRLKSSCK RHPLY 316NSKDPKACCVPTELSAPSP C67-C71^(a), C8-C43^(a), 6.17 ±1.04LYLDENEKPVLKNYQDMV C9-C41^(a), C109-C109^(b) VHGCGCRVDFKDVGWNDHAVAPPGYHAFYCHGECPF PLADHLNSDNHAIVQTKVN SVGQAKRKGYKRLKSSCK RHPLY 316NSKDPKACCVPTELSAPSP C67-C71^(a), C8-C43^(b), 5.78 ±1.18LYLDENEKPVLKNYQDMV C9-C41^(b), C109-C109^(b) VHGCGCRVDFKDVGWNDHAVAPPGYHAFYCHGECPF PLADHLNSDNHAIVQTKVN SVGQAKRKGYKRLKSSCK RHPLY 324AQAKHKQRKRLKSSCKRH C15-C15^(b), C79- 8.42 ±1.59 PLYVDFKDVGWNDHAVAPC114^(a), C80-C112^(a), PGYHAFYCHGECPFPLADH C44-C48^(a)LNSDNHAIVQTKVNSVNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 324AQAKHKQRKRLKSSCKRH C79-C80^(a) 29.33 ±2.10 PLYVDFKDVGWNDHAVAPPGYHAFYCHGECPFPLADH LNSDNHAIVQTKVNSVNSK IPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGC GCR 324 AQAKHKQRKRLKSSCKRH C80-C114^(a), C79- 7.22±2.15 PLYVDFKDVGWNDHAVAP C112^(a), C44-C48^(a) PGYHAFYCHGECPFPLADHLNSDNHAIVQTKVNSVNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 324AQAKHKQRKRLKSSCKRH C15-C15^(b), C80- 9.11 ±1.77 PLYVDFKDVGWNDHAVAPC114^(b), C79-C112^(b), PGYHAFYCHGECPFPLADH C44-C48^(a)LNSDNHAIVQTKVNSVNSK IPKACCVPTELSAISMLYLD ENEKVVLKNYQDMVVEGC GCR 340AQAKHKQRKRLKSSCKRH C15-C15^(b), C29-C62^(a), 5.77 ±1.17PLYNSKDPKACCVPTELSA C30-C64^(a), C88-C92^(a) PSPLYLDENEKPVLKNYQDMVVHGCGCRVDFKDVGW NDHAVAPPGYHAFYCHGE CPFPLADHLNSDNHAIVQT KVNSV 340AQAKHKQRKRLKSSCKRH C15-C29^(a) 30.05 ±2.63 PLYNSKDPKACCVPTELSAPSPLYLDENEKPVLKNYQD MVVHGCGCRVDFKDVGW NDHAVAPPGYHAFYCHGECPFPLADHLNSDNHAIVQT KVNSV 340 AQAKHKQRKRLKSSCKRHC15-C15^(b), C29-C64^(a), 7.19 ±1.95 PLYNSKDPKACCVPTELSAC30-C62^(a), C88-C92^(a) PSPLYLDENEKPVLKNYQD MVVHGCGCRVDFKDVGWNDHAVAPPGYHAFYCHGE CPFPLADHLNSDNHAIVQT KVNSV 340 AQAKHKQRKRLKSSCKRHC15-C15^(b), C29-C64^(b), 6.37 ±1.55 PLYNSKDPKACCVPTELSAC30-C62^(b), C88-C92^(a) PSPLYLDENEKPVLKNYQD MVVHGCGCRVDFKDVGWNDHAVAPPGYHAFYCHGE CPFPLADHLNSDNHAIVQT KVNSV 332 GQAKRKGYKRLKSSCKRHC15-C15^(b), C80- 5.98 ±1.11 PLYVDFSDVGWNDWIVAP C114^(a), C79-C112^(a),PGYHAFYCHGECPFPLADH C44-C48^(a) LNSTNHAIVQTLVNSVNSK DPKACCVPTELSAPSPLYLDENEKPVLKNYQDMVVHG CGCR 332 GQAKRKGYKRLKSSCKRH C79-C80^(a) 28.54 ±2.36PLYVDFSDVGWNDWIVAP PGYHAFYCHGECPFPLADH LNSTNHAIVQTLVNSVNSKDPKACCVPTELSAPSPLYL DENEKPVLKNYQDMVVHG CGCR 332 GQAKRKGYKRLKSSCKRHC15-C15^(b), C80- 7.18 ±1.84 PLYVDFSDVGWNDWIVAP C112^(a), C79-C114^(a),PGYHAFYCHGECPFPLADH C44-C48^(a) LNSTNHAIVQTLVNSVNSK DPKACCVPTELSAPSPLYLDENEKPVLKNYQDMVVHG CGCR 332 GQAKRKGYKRLKSSCKRH C15-C15^(b), C80- 7.42±1.94 PLYVDFSDVGWNDWIVAP C114^(b), C79-C112^(b), PGYHAFYCHGECPFPLADHC44-C48^(a) LNSTNHAIVQTLVNSVNSK DPKACCVPTELSAPSPLYL DENEKPVLKNYQDMVVHGCGCR 348 GQAKRKGYKRLKSSCKRH C15-C15^(b), C29-C62^(a), 5.10 ±1.07PLYNSKIPKACCVPTELSAI C30-C64^(a), C88-C92^(a) SMLYLDENEKVVLKNYQDMVVEGCGCRVDFSDVGW NDWIVAPPGYHAFYCHGE CPFPLADHLNSTNHAIVQT LVNSV 348GQAKRKGYKRLKSSCKRH C29-C30^(a) 29.09 ±3.33 PLYNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQD MVVEGCGCRVDFSDVGW NDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQT LVNSV 348 GQAKRKGYKRLKSSCKRHC15-C15^(b), C29-C64^(a), 6.77 ±1.92 PLYNSKIPKACCVPTELSAIC30-C62^(a), C88-C92^(a) SMLYLDENEKVVLKNYQD MVVEGCGCRVDFSDVGWNDWIVAPPGYHAFYCHGE CPFPLADHLNSTNHAIVQT LVNSV 348 GQAKRKGYKRLKSSCKRHC15-C15^(b), C29-C62^(b), 6.31 ±1.45 PLYNSKIPKACCVPTELSAIC30-C64^(b), C88-C92^(a) SMLYLDENEKVVLKNYQD MVVEGCGCRVDFSDVGWNDWIVAPPGYHAFYCHGE CPFPLADHLNSTNHAIVQT LVNSV ^(a)Intramoleculardisulfide bond connection. bIntermolecular disulfide bond connection fordimerization.

As shown in TABLE 4, the disulfide bond between different cysteinelocations affects affinity constant (K_(D)) values. In addition, thedata show that a disulfide bond between two recombinant polypeptides ina dimer significantly reduces the K_(D) values. In other words,dimerization might assist in vitro molecular bonding between the dimersof recombinant polypeptides and the ActRIIBecd.

Some recombinant polypeptides were observed to spontaneously formdimeric proteins as shown in Table 4. All of the dimeric proteins couldbe fractionated out of the recombinant polypeptide by gel filtrationdescribed in Example 4B. In this embodiment, the dimeric proteins werehomodimeric protein because their monomer were the same. In the otherembodiments, the dimeric proteins could be heterodimeric protein if thestably transformed E. coli cells as described in Example 4 areco-expressed by two different recombinant polypeptides selected from thegroup of SEQ ID Nos: 260, 268, 276, 284, 292, 300, 308, 316, 324, 332,340, and 348.

Example 8: Alkaline Phosphatase Bioactivity Assay

The ability of the recombinant polypeptides to bind to cellularreceptors and induce signal transduction pathways was investigated usingan assay for alkaline phosphatase induction in C2C12 cells, which hasbeen described. See, e.g., Peel et al. J Craniofacial Surg. 2003;14:284-291 and Hu et al. Growth Factors 2004; 22:29033.

C2C12 cells (ATCC accession number CRL-1772, Manassas, Va.) werepassaged before confluent and resuspended at 1×10⁵ cells/mL in DMEMsupplemented with 10% heat-inactivated fetal bovine serum. 100 μL ofcell suspension was seeded per well of a 96 well tissue culture plate(Corning, Cat #3595). Aliquots of serial diluted standard and testsample were added and the cultures maintained at 37° C. and 5% CO₂. Testsamples included conditioned media, purified recombinant polypeptide,and as a positive control a commercially available purified recombinanthuman BMP-2 “rhBMP-2” (R&D Systems, Minneapolis, USA). rhBMP-2 has beenshown to play an important role in the development of bone and cartilageby, for example, Mundy GR., et al. (2004, Growth Factors. 22 (4):233-41). Negative control cultures (cultured in media without addedsample or rhBMP-2) were cultured for 2 to 7 days. Medium was changedevery two days.

At harvest cultures were rinsed with normal saline (0.90% NaCl, pH 7.4)and discard the rinsed saline. 50 μL extraction solution (Takara Bio,catalogue #MK301) was added to those cultures and then sonicated at roomtemperature for 10 minutes. The lysate was assayed for alkalinephosphatase (ALP) by monitoring the hydrolysis of nitrophenol phosphatein alkaline buffer (Sigma-Aldrich, St. Louis Mo., catalog P5899) asdescribed in Peel et al. J Craniofacial Surg. 2003; 14:284-291 or byusing the TRACP & ALP assay kit (Takara Bio, catalogue #MK301) accordingto manufacturer's instructions. ALP activity was determined by recordingabsorbance at 405 nm. An activity score was calculated by mean ALPactivity of duplicate samples. Serial diluted samples and its relevantactivity score were diagramed by 4-parameter curve fit so as tocalculate the concentration EC₅₀ of each recombinant polypeptide. Datais shown in TABLE 5. In some embodiments, the ALP activity the cellularprotein content in each well is normalized by using the Coomasie(Bradford) Protein Assay (Pierce Biotechnology Inc., catalogue #23200).The normalized ALP activity for each sample is calculated by dividingthe ALP activity per well by the protein content per well.

In another embodiment, alkaline phosphatase assay described by Katagiri,T., et al. (1990, Biochem. Biophys. Res. Commun. 172, 295-299) isperformed. Mouse fibroblast cells from the line C3H10T1/2 in BME-Earlemedium plus 10% fetal calf serum are incubated at 1×10⁵ cells/mL in 1-mLaliquots in a 24-well plate for 24 h at 37° C. and 10% CO₂. Afterremoval of the supernatant, 1 mL fresh medium is added with variousconcentrations of sample. After a further cultivation for 4 days, cellsare lysed in 0.2 mL buffer (0.1 M glycerol, pH 9.6, 1% NP-40, 1 mMMgCl₂, 1 mM ZnCl₂) and alkaline phosphatase activity is determined in 50μL aliquots of the cleared lysate using 150 μL 0.3 mM p-nitrophenylphosphate in the pH 9.6 buffer as substrate. Absorbance at 405 nm isrecorded after 20 min incubation at 37° C. The activity is related tothe protein content (BCA protein assay, Pierce Chemical Co.) in eachsample.

TABLE 5 Identified EC₅₀ Mean K_(D) SEQ ID NO Cysteine Pairs [nM] [nM]188 C15-C44^(a) NA 42.32 188 C44-C48^(a) 28.4 21.47 194 C23-C65^(a) NA45.98 194 C23-C27^(a) NA 18.14 200 C23-C27^(a), C58-C91^(a), C59- 31.218.62 C93^(a) 200 C23-C27^(a), C58-C93^(a), C59- NA 20.13 C91^(a) 200C23-C27^(a), C58-C91^(b), C59- 35.1 19.75 C93^(b) 200 C23-C58^(a) NA47.62 206 C67-C71^(a), C8-C41^(a), C9-C43^(a) 37.6 15.49 206 C8-C71^(a)NA 49.66 206 C67-C71^(a), C8-C43^(a), C9-C41^(a) NA 17.73 206C67-C71^(a), C8-C43^(b), C9-C41^(b) 40.1 16.97 212 C59-C59^(b),C8-C41^(a), C9-C43^(a) 35.1 18.13 212 C59-C59^(b), C8-C43^(a),C9-C41^(a) 36.7 19.34 212 C41-C59^(a) NA 44.33 218 C15-C15^(b),C29-C62^(a), C30- 33.9 16.22 C64^(a) 218 C15-C15^(b), C29-C64^(a), C30-37.6 18.42 C62^(a) 218 C15-C30^(a) NA 40.89 224 C44-C48^(a) NA 21.47 224C15-C44^(a) NA 48.93 230 C23-C27^(a) NA 17.94 230 C27-C65^(a) NA 52.23236 C59-C59^(b), C8-C41^(a), C9-C43^(a) 28.5 16.21 236 C41-C59^(a) NA49.51 236 C59-C59^(b), C8-C43^(a), C9-C41^(a) 29.7 17.05 242C15-C15^(b), C29-C62^(a), C30- 30.1 17.78 C64^(a) 242 C29-C30^(a) NA48.66 242 C15-C15^(b), C29-C64^(a), C30- 33.9 18.11 C62^(a) 248C67-C71^(a), C8-C41^(a), C9-C43^(a) 29.4 18.52 248 C41-C43^(a) NA 47.81248 C67-C71^(a), C8-C43^(a), C9-C41^(a) NA 19.98 248 C67-C71^(a),C8-C43^(b), C9-C41^(b) 30.5 19.25 254 C23-C27^(a), C58-C91^(a), C59-31.4 17.55 C93^(a) 254 C23-C27^(a), C58-C93^(a), C59- NA 19.06 C91^(a)254 C23-C58^(a) NA 44.43 254 C23-C27^(a), C58-C91^(b), C59- 34.4 18.73C93^(b) 260 C15-C80^(a), C44-C112^(a), C48- 14.1 6.56 C114^(a),C79-C79^(b) 260 C79-C80^(a) 67.8 29.14 260 C15-C15^(b), C79-C114^(a),C80-  1.1 7.64 C112^(a), C44-C48^(a) 260 C80-C114^(a), C79-C112^(a),C44- 19.4 8.31 C48^(a) 260 C15-C15^(b), C80-C114^(a), C79-  1.3 7.45C112^(a), C44-C48^(a) 260 C79-C114^(a), C80-C112^(a), C44- 15.6 6.17C48^(a) 260 C44-C48^(a)  9.8 8.04 260 C15-C15^(b), C80-C114^(b), C79- 1.0 4.04 C112^(b), C44-C48^(a) 260 C15-C15^(b), C79-C114^(b), C80-  1.94.03 C112^(b), C44-C48^(a) 260 C80-C114^(b), C79-C112^(b), C44- 13.46.89 C48^(a) 260 C79-C114^(b), C80-C112^(b), C44- 10.5 6.05 C48^(a) 268C23-C27^(a), C65-C65^(b), C79- 18.4 7.21 C112^(a), C80-C114^(a) 268C23-C79^(a) 71.1 29.57 268 C23-C27^(a), C65-C65^(b), C79- 20.4 8.74C114^(a), C80-C112^(a) 268 C23-C27^(a), C65-C65^(b), C79- 18.6 7.96C112^(b), C80-C114^(b) 268 C23-C27^(a), C65-C65^(b), C79- 24.5 8.03C114^(b), C80-C112^(b) 268 C23-C27^(a) 23.4 8.41 276 C67-C71^(a),C8-C41^(a), C9- 21.9 5.71 C43^(a), C109-C109^(b) 276 C8-C9^(a) 130.1 27.93 276 C67-C71^(a), C8-C43^(a), C9- 25.7 7.66 C41^(a), C109-C109^(b)276 C67-C71^(a), C8-C43^(b), C9- 31.7 6.31 C41^(b), C109-C109^(b) 276C67-C71^(a), C8-C41^(b), C9- 26.3 8.13 C43^(b), C109-C109^(b) 276C67-C71^(a) 22.5 7.41 284 C59-C59^(b), C8-C41^(a), C9- 27.1 4.89C43^(a), C88-C92^(a) 284 C8-C9^(a) 83.9 31.79 284 C59-C59^(b),C8-C43^(a), C9- 36.7 6.88 C41^(a), C88-C92^(a) 284 C59-C59^(b),C8-C41^(b), C9- 33.9 5.79 C43^(b), C88-C92^(a) 284 C59-C59^(b),C8-C43^(b), C9- 19.5 6.91 C41^(b), C88-C92^(a) 284 C88-C92^(a) 25.9 8.14292 C15-C80^(a), C44-C112^(a), C48- 10.7 4.77 C114^(a), C79-C79^(b) 292C79-C80^(a) 89.4 28.99 292 C15-C15^(b), C79-C114^(a), C80- 22.5 5.81C112^(a), C44-C48^(a) 292 C80-C114^(a), C79-C112^(a), C44- 19.8 6.19C48^(a) 292 C80-C114^(b), C79-C112^(b), C44- 22.3 5.54 C48^(a) 292C44-C48^(a) 27.5 7.81 300 C23-C27^(a), C65-C65^(b), C79- 17.6 5.14C112^(a), C80-C114^(a) 300 C23-C79^(a) 86.1 32.69 300 C23-C27^(a),C65-C65^(b), C79- 12.1 7.37 C114^(a), C80-C112^(a) 300 C23-C27^(a),C65-C65^(b), C79- 27.7 6.44 C112^(b), C80-C114^(b) 300 C23-C27^(a) 16.58.92 308 C59-C59^(b), C8-C41^(a), C9- 33.9 5.98 C43^(a), C88-C92^(a) 308C8-C9^(a) 77.4 31.22 308 C59-C59^(b), C8-C43^(a), C9- 20.4 6.11 C41^(a),C88-C92^(a) 308 C59-C59^(b), C8-C41^(b), C9- 36.7 6.73 C43^(b),C88-C92^(a) 308 C88-C92^(a) 38.2 7.8 308 C8-C43^(a), C9-C41^(a),C88-C92^(a) 21.7 10.9 316 C67-C71^(a), C8-C41^(a), C9- 21.0 5.34C43^(a), C109-C109^(b) 316 C8-C67^(a) 109.1  28.91 316 C67-C71^(a),C8-C43^(a), C9- 41.5 6.17 C41^(a), C109-C109^(b) 316 C67-C71^(a),C8-C43^(b), C9- 18.5 5.78 C41^(b), C109-C109^(b) 316 C67-C71^(a) 33.94.67 324 C15-C15^(b), C79-C114^(a), C80- 17.7 8.42 C112^(a), C44-C48^(a)324 C79-C80^(a) 88.7 29.33 324 C80-C114^(a), C79-C112^(a), C44- 27.17.22 C48^(a) 324 C15-C15^(b), C80-C114^(b), C79- 37.6 9.11 C112^(b),C44-C48^(a) 324 C44-C48^(a) 29.5 8.75 340 C15-C15^(b), C29-C62^(a), C30-18.9 5.77 C64^(a), C88-C92^(a) 340 C15-C29^(a) 78.4 30.05 340C15-C15^(b), C29-C64^(a), C30- 21.2 7.19 C62^(a), C88-C92^(a) 340C15-C15^(b), C29-C64^(b), C30- 26.7 6.37 C62^(b), C88-C92^(a) 340C88-C92^(a) 36.7 5.64 332 C15-C15^(b), C80-C114^(a), C79- 22.4 5.98C112^(a), C44-C48^(a) 332 C79-C80^(a) 102.3  28.54 332 C15-C15^(b),C80-C112^(a), C79- 14.9 7.18 C114^(a), C44-C48^(a) 332 C15-C15^(b),C80-C114^(b), C79- 33.6 7.42 C112^(b), C44-C48^(a) 348 C15-C15^(b),C29-C62^(a), C30- 20.5 5.10 C64^(a), C88-C92^(a) 348 C29-C30^(a) 90.329.09 348 C15-C15^(b), C29-C64^(a), C30- 18.9 6.77 C62^(a), C88-C92^(a)348 C15-C15^(b), C29-C62^(b), C30- 35.6 6.31 C64^(b), C88-C92^(a) 348C88-C92^(a) 27.6 4.51 rhBMP-2 NA 45.2 NA NA: not yet analyzed^(a)Intramolecular disulfide bond connection. ^(b)Intermoleculardisulfide bond connection for dimerization.

As shown by TABLE 5, most of the recombinant polypeptides with certaindisulfide connections have a value of EC₅₀ lower than that of rhBMP-2.In other words, most of the recombinant polypeptides with certaindisulfide connections were able to induce a signal transduction pathwayrelated to bone or cartilage formation or osteogenesis.

Example 9: In Vivo Osteoinductive Activity

Osteoinductive activity of a homodimeric protein including tworecombinant polypeptides produced according to Example 6 (i.e., SEQ IDNO: 260, including intramolecular disulfide bond C44-C48) and porousbeta-tricalcium phosphate (β-TCP) as a carrier material was evaluated inulnar shaft defects in rabbits. The calcium phosphate carrier has acalcium to phosphate ratio of about 0.4 to about 1.65.

20 mm-sized circumferential defects were created in the shaft ofsurgically exposed right and left ulnae in each of 40 female rabbits(strain NZW, Japan SLC, Inc.). Briefly, combined anesthesia was carriedout with ketamine hydrochloride (Ketalar, Daiichi Sankyo Co., Ltd.) andxylazine (Selactar 2% injection solution, Bayer Medical Co., Ltd.) at acombined rate of 3:1. The same solution was used for additionalanesthesia in long operations. Before operating, Flumarin (flomoxefsodium, Shionogi & Co., Ltd.) was administered subcutaneously as anantibiotic agent. Fur in the general region of the forearm was shavedwith an electric shaver and disinfected with Hibitane alcohol (0.5%chlorhexidine gluconate-ethanol solution, Sumitomo Dainippon Pharma,Co., Ltd.). A longitudinal incision was made on each caudomedial part oflimb over the ulna. Muscle tissue was lifted to expose the ulna. A markwas made with a scalpel 25 mm from the hand joint of the exposed ulna.Proper holes were drilled longitudinally and vertically at the markusing a 15 mm diameter drill, paying close attention not to break thebone. The bone was split with a luer bone rongeurs. A mark was also madeat 20 mm away in the proximal direction, and split similarly. Whensplit, the ulna was covered with periosteum, which was then removed, andthe bone fragments were thoroughly cleaned with saline.

Each ulna then received an implant or no implant according to one ofGroups A-G, shown in Table 6, below. Groups A-D ulnae received a singleimplant of β-TCP carrying a specified amount of homodimeric protein.Group E ulnae received a single implant of β-TCP alone, without anyhomodimeric protein. Group F ulnae received a single implant of a boneautograft. Group G ulnae received no implant. Afterwards, muscle anddermal tissues were promptly sutured.

TABLE 6 Homodimeric protein β-TCP Homodimeric protein dose per β-TCPGroup (mg) (μg) (mg/g) A 200 2 0.01 B 200 6 0.03 C 200 20 0.1 D 200 600.3 E 200 0 0 F Autograft (iliac bone fragments): 0.55 g on average (noβ-TCP or homodimeric protein) G Defect only (no β-TCP or homodimericprotein)

β-TCP as used in Groups A-E was in the form of 1-3 mm granules with aporosity of 75% and a pore diameter of 50-350 μm (Superpore™, pentax,“HOYA” Bone Graft Substitute, Japan).

In certain embodiments, β-TCP as used in Groups A-E is in the form of1-3 mm granules with a porosity of 70% or more and a pore diameter of300-600 μm (“Wiltrom” Osteocera Bone Graft Substitute, Wiltrom Co.,Ltd., Taiwan, R.O.C.).

Homodimeric protein including recombinant polypeptides (i.e., SEQ ID NO:260) in Groups A-D was prepared from frozen lots immediately beforeimplantation for each animal using 0.5 mM hydrochloric acid (standardsolution diluted with injection solvent (Otsuka Pharmaceutical Co.,Ltd.)). Fluid volume was set at 180 μl for unilateral implantation andwas dropped evenly across 200 mg of β-TCP granules in a sterilized Petridish. When the fluid was completely dropped, the β-TCP granules weregently stirred with a spatula, allowed to sit for more than 15 minutesat room temperature, and then implanted.

For Group F, autograft bone was obtained from either the right or leftwing of the ilium using luer bone rongeurs. Bones were processed intochips, and the same amount of bone was implanted as the amounts inGroups A-E.

X-Ray Evaluation

Lateral and frontal X-ray images (i.e., radiographs) were takenimmediately after implantation and once every two weeks thereafter until8 weeks after implantation. The condition of the implanted sites and thedegree of bone formation were evaluated using the radiographs.Representative examples of X-ray images for each group are shown in FIG.1A (Groups A-D) and FIG. 1B (Groups E-G).

At 2 weeks, contrast of granules of grafting material and the boundarywith the recipient bed were clearly seen in all groups. At 4 weeks, TCPgranules became unclear in the homodimeric protein groups (i.e., GroupsA-D), showing absorption of the granules and progress of bone formation.The boundaries between the implanted site and the recipient bed wereunclear in some samples in Group C and in Group D with high dose of thehomodimeric protein. At 6 weeks, the boundary between the implanted siteand the recipient bed became unclear in Group B. Improved continuity inthe recipient bed and formation of the bone cortex were observed in somesamples in Group C and in Group D. At 8 weeks, the boundary at therecipient bed became even more unclear in Groups A and B. Continuity inthe recipient bed and formation of bone cortex improved in Group C.Reconstruction of the region of the ulnar defect was observed in GroupD, as shown in the image at 6 weeks.

In Group E with TCP alone, bone formation in the recipient bed wasobserved over time. However, remaining TCP granules were clearly seeneven at 8 weeks, showing insufficient bone formation at the implantationsite and poor continuity in the recipient bed. Thus, repair of defectsin Group E was still incomplete at 8 weeks.

In Group F with autograft, progress of bone formation was observed overtime, and fusion with the recipient bed was achieved at 8 weeks.However, formation was not uniform.

In Group G with only the defect and no implant, slight bone formation inthe radius was observed at 8 weeks without any other repair of thedefect.

Computerized Tomography (CT) Scanning

Axial orientation was performed at 1 mm intervals using CT scanning (GEYokogawa Medical Systems Ltd.) immediately, at 4 weeks, and at 8 weeksafter implantation. Images were mainly taken at the implant sites.Change in cross-sectional images over time at the center of theimplanted sites are shown for representative examples in FIG. 2A (GroupsA-D) and FIG. 2B (Groups E-G).

In Groups A to D with homodimeric protein, the granules observedimmediately after implantation were partially degraded in thecross-sectional image at 4 weeks, suggesting bone formation. In Group Dhaving a dose of 60 μg, further progress in bone formation was observed,and the formation of bone-marrow cavities in some samples was observed.At 8 weeks, progress in the formation of bone-marrow cavities and bonecortex was observed in the images for groups with doses over 6 μg. InGroup E with only TCP, an agglomerated mass of granules remained even at8 weeks. In Group F with autograft, formation of bone-marrow cavitieswas observed at 8 weeks, as in the remodeling process. In Group G withonly defect, only slight bone formation was observed.

Torsional Strength Test

The grafting materials were removed from rabbits that were euthanized 8weeks after implantation, and a torsional strength test was conductedfor each group on the ulnae samples from which the radii were separated.858 Mini Bionix II (MTS Systems Corporation) was used for the test. Thetest was conducted on a 50 mm-long area, namely, the 20 mm-longreconstructed area in the ulnar shaft at the center, and the 15 mm-longareas on both the proximal and distal sides of the reconstructed area.The edges of each side were fixed with dental resin. The resin partswere chucked in a measurement equipment. The left ulna was turnedcounterclockwise and the right ulna clockwise at a rotation rate of30°/min, in order to determine maximum torque at failure. The separatelyobtained ulnae of healthy rabbits were also examined and compared. Thesehealthy ulnae were obtained from Japanese white rabbits, a differenttype than those used in Groups A-E. The Japanese white rabbits were,however, the same age and gender as for Groups A-E at the time ofeuthanization, namely 26 weeks old and female.

The maximum torque of each group obtained by the torsional strength testis shown in FIG. 3. In Groups A-D with homodimeric protein, maximumtorque was dose-dependently high.

Significantly high values were seen in Groups A-D with a dose of 2 μgand over of homodimeric protein as compared to Group E with TCP alone.

Significantly high values were also seen in Groups B-D with a dose of 6μg and over of homodimeric protein as compared to Group G with defectonly.

No significant difference was observed among groups with an intact ulna,autograft, or homodimeric protein.

Due to insufficient bone formation in Groups E and G, it was difficultto ensure the support in some samples when the radii were separated.Therefore, only 2 samples from Group E and 4 sample from Group G wereused in the test, while 6 samples were used from each of Groups A-D andF.

Table 7 below shows a comparison of test conditions and results betweenthis study and the evaluation of CHO-derived BMP-2 in Kokubo et al.,Biomaterials 24:1643-1651 (2003), using the same animal model. Comparedto the report by Kokubo et al., this study was conducted under moredifficult conditions, such as a larger bone defect, a smaller dose ofactive agent, and a shorter duration of implantation prior to torsionalstrength testing. However, ulnae were shown to be successfully repairedin this study, and maximum torque in this study was remarkably similar.

TABLE 7 Size of Length of Source defect Weeks after torsion of testChemical agent Maximum of data Carrier (mm) implantation specimen (mm)dose torque Kokubo PGS* 15 16 45 100 μg BMP-2 0.307 et al. 400 μg BMP-20.365 1000 μg BMP-2 0.413 This Study TCP 20 8 50 2 μg h.p.{circumflexover ( )} 0.262 granule 6 μg h.p. 0.375 20 μg h.p. 0.392 60 μg h.p.0.400 *PGS: PLGA-coated Gelatin Sponge {circumflex over ( )}h.p.:homodimeric protein

Histological Evaluation

Specimens were prepared for all animals in 8 week and 4 week groups.Tissue obtained at the time of necropsy were preserved in 4%paraformaldehyde solution and decalcified with 10% EDTA. The tissue wasthen paraffin-embedded. Thinly-sliced samples were prepared on the planerunning parallel to the long axis of the radius, hematoxylin and eosin(HE) stained, and histologically evaluated. Conditions of bone formationand fusion to the recipient bed were determined.

In Groups A to D with homodimeric protein, bone formation progressed toa trabecular pattern at 4 weeks. Active bone formation was observed insamples with a high dose of homodimeric protein. A significantly largeamount of new bone and angiogenesis were observed in Group D with a doseof 60 μg. Remaining material was observed in some samples in Groups Aand B with a low dose, while almost none was observed in Groups C and D.In Groups A and B, cartilage formation was observed in some samples nearthe boundary with the recipient bed. In all samples, the recipient bedwas directly connected to the newly formed bone in a trabecular pattern.At 8 weeks, a trabecular pattern and remaining materials were stillobserved in Group A with a dose of 2 μg. Cartilage was also observednear the boundary with the recipient bed. Even though remodeling wasinsufficient, progress in bone formation was observed. The formation ofbone cortex in the radii was observed in some samples. In Groups B to Dwith doses over 6 μg, bone cortex and bone marrow were being formed byremodeling. The progress was more significant in higher dose groups.Continuity in the recipient site also increased.

In Group E with TCP alone, bone formation on the grafting materials wasobserved in the radii, but remaining materials were still clearly seeneven at 8 weeks, showing insufficient bone formation on the shaft andpoor continuity.

In Group F with autograft, good bone formation on the grafted bonefragments was seen at 4 weeks, and new bone was in contact with therecipient bed. Cartilage formation was seen near the boundary with therecipient bed. At 8 weeks, progress in remodeling of new bone andformation of bone cortex were observed, but remaining grafted bonefragments were still observed.

In Group G with only defect, bone formation was observed only in theradii, and repair of the defect was not achieved.

In the embodiment, a method of promoting healing of a long-bone fracturein a subject in need of such treatment was provided. The method includespreparing a composition including the homodimeric protein homogeneouslyentrained within a slow release biodegradable calcium phosphate carrier(e.g., β-TCP) that hardens so as to impermeable to efflux of thehomodimeric protein in vivo sufficiently that the long-bone fracturehealing is confined to the volume of the calcium phosphate carrier andimplanting the composition at a location where the long-bone fractureoccurs, wherein the homodimeric protein is in an amount of from about0.03 mg/g to about 3.2 mg/g of the calcium phosphate carrier.

Example 10: In Vivo Ovine Posterolateral Fusion Study

Osteoinductive activity of a homodimeric protein including a recombinantpolypeptide (Rcp) produced according to Example 6 (i.e., SEQ ID NO: 260,including intramolecular disulfide bonds C44-C48, C80-C112 and C79-C114)and porous beta-tricalcium phosphate (β-TCP) as a carrier material wasevaluated in ovine posterolateral fusion model. The calcium phosphatecarrier has a calcium to phosphate ratio of about 1.2 to about 1.8.

The ovine posterolateral fusion model used sheep sedated with Zoletil(8-12 mg/kg, IM), and gassed down with isofluorane (2%) and oxygen (2litres per minute). An endotracheal tube was inserted and the animalventilated, anesthesia was maintained using isofluorane (2 to 3%) andoxygen (2-4 litres per minute). Antibiotics (Keflin®: 1 gm IV;Benacillin 5 ml IM) were given. Carprofen (an NSAID, 4 ml IM) andTemgesic® (Burprenorphine 0.324 mg SC) were injected prior to surgery.Crystalloid fluids (Hartmann's solution) were given intravenously at 4to 10 ml/kg/h prior to and during the surgery as required.

A 15-cm midline incision was made parallel to the lumbar transverseprocess at the L3-L4 level. Blunt retroperitoneal dissection exposed theanterolateral aspect of the lumbar spine. This left the diaphragmundisturbed. The soft tissues were retracted. A pneumatic burr (MidasRex) was used to decorticate the transverse processes (15 mm lateral)and adjacent vertebral body between the levels in all animals.

The graft materials were placed between the decorticated surfaces of thetransverse processes and the vertebral bodies (paraspinal bed) accordingto one of Groups 1-6, shown in Table 8, below. Groups 1-3 received asingle implant of β-TCP carrying a specified amount of homodimericprotein. Group 4 received a single implant of β-TCP alone, without anyhomodimeric protein. Group 6 received a single implant of absorbablecollagen sponge (ACS) with a specified amount of rhBMP-2, awell-established osteoinductive factor as positive control. Group 5received a single implant of bone autograft. Autografts were harvestedfrom the iliac crests in the autograft group animals. The bones weremorcelized using a rongeur and 5.0 g of autograft bone used for eachside of the fusion. The incisions were closed with 2-0 absorbable sutureand the skin approximated using 3-0 suture.

TABLE 8 Homodimeric protein Group/No. (mg/site) Carrier Fixation Timepoint Sample Size 1 h.p.{circumflex over ( )} High 10.5 3.5 g β-TCP None12 wks 6 2 h.p.{circumflex over ( )} Middle 3.5 3.5 g β-TCP None 12 wks6 3 h.p.{circumflex over ( )} Low 1.05 3.5 g β-TCP None 12 wks 6 4 NoneN/A 3.5 g β-TCP None 12 wks 6 5 None N/A Autograft None 12 wks 6 6Infuse ® + 3.15 rh- ACS 4 cm³ None 12 wks 6 Mastergraft ® BMP-2Mastergraft ® mg/site 5 cm³ {circumflex over ( )}h.p.: homodimericprotein

β-TCP as used in Groups 1-4 was in the form of 2-4 mm granules with aporosity of 70% and a pore diameter of 50-350 μm (Superpore™, pentax,“HOYA” Bone Graft Substitute, Japan).

In certain embodiments, β-TCP as used in Groups 1-4 is in the form of2-8 mm granules with a porosity of 65% or more and a pore diameter of250-730 μm (“Wiltrom” Osteocera Bone Graft Substitute, Wiltrom Co.,Ltd., Taiwan, R.O.C.).

In Group 1, homodimeric protein including Rcp (i.e., SEQ ID NO: 260)stock solution H was prepared by adding 2 ml injection water to each ofvials with 10 mg homodimeric protein. The stock solution H was mixedwith injection water by 3:1 on volume ratio to form Homodimeric ProteinHigh Dose Solution (containing 10.5 mg homodimeric protein in 2.8 mlsolution). 2.8 ml Homodimeric Protein High Dose Solution was deliveredby dropping evenly into 3.5 g β-TCP granule.

In Group 2, homodimeric protein stock solution ML (2.5 mg/ml) wasprepared by adding 4 ml injection water to each of vial with 10 mghomodimeric protein. The stock solution ML was mixed with injectionwater by 1:1 on volume ratio to form homodimeric protein Middle DoseSolution (containing 3.5 mg homodimeric protein in 2.8 ml solution). 2.8ml Homodimeric Protein Middle Dose Solution was delivered by droppingevenly into 3.5 g β-TCP granule.

In Group 3, homodimeric protein stock solution ML (2.5 mg/ml) wasprepared by adding 4 ml injection water to each of vial with 10 mghomodimeric protein. The stock solution ML was mixed with injectionwater by 17:3 on volume ratio to form homodimeric protein Low DoseSolution (containing 1.05 mg homodimeric protein in 2.8 ml solution).2.8 ml homodimeric protein Low Dose Solution was delivered by droppingevenly into 3.5 g β-TCP granule.

Group 6 was conducted to compare commercial product Infuse® andMastergraft® as graft materials, both are distributed by Medtronic.Infuse® consisted of rh-BMP-2 prepared by CHO expression system andabsorbable collagen sponge (“ACS”). Mastergraft® is granular calciumphosphate bone substitute consisting of 85% β-TCP and 15%Hydroxyapatite. Application of Infuse® combined with Mastergraft® onposterolateral lumber fusion showed efficacy and was reported by E.Dawson et. al., on the clinical study with evidence level 2 (J BoneJoint Surg Am. 2009; 91: 1604-13). Graft material for Group 6 consistedof 3.15 mg rh-BMP-2, 4 cc ACS and 5 cc Mastergraft® per site, andprocedure of graft material preparation were followed as E. Dawson'sreport. Lot number of Infuse® and Mastergraft® were recorded onoperation record.

The animals were monitored daily for the first 7 days following surgeryand observations recorded on post-operative monitoring sheets for eachanimal.

Posteroanterior radiographs of all animals were taken at 4 weeks. Theanimals were sedated with Zoletil® (8-12 mg/kg, IM), and gassed downwith Isofluorane (2%) and oxygen (2 litres per minute). The radiographswere used to compare to the post-operative X-rays for the presence ofnew bone and absorption of the TCP material. At 12 weeks post surgeryall animals were sacrificed via lethal cardiac injection of Lethobarb.

To monitor the time of bone formation, three different fluorochromeswere intravenously injected at three time points as indicated in theTable 9 below.

TABLE 9 The date and dosage of fluorochrome used* Alizarin complexoneCalcien Engemycin ® Supplier Sigma Sigma Schering Plough Solvent 1.4%NaHCO₃ 1.4% NaHCO₃ Physiological saline pH 6-8 6-8 6-8 Dosage (mg/kg 28mg/kg 10 mg/kg 20 mg/kg body weight) (=2 ml/kg) (=2 ml/kg) (=2 ml/kg)Time to inject 6 weeks 8 weeks 10 weeks after *All filtered through 0.22μm filters before use

X-Ray Evaluation

The lumbar spines (L1-L6) were harvested and photographed using adigital camera. The harvested spines were faxitroned using a Faxitron®Machine (settings 24 kV for 45 seconds). Digital radiographs were takenin the posterior-anterior (PA) were graded for evidence of new boneformation and fusion by three blinded observers on the right and leftsides. A qualitative grading system was used to assess the radiographs(Table 10). Fusion was assessed based on a continuous pattern of bonefrom one transverse process to the next level (0=non-continuous,1-continuous). The amount of bone between the transverse processes oneach side of the fusion masses was graded based on percentage asoutlined in Table 10. The amount of TCP resorption was noted based on acomparison to time 0 radiographs with the same amount of material.

TABLE 10 Radiographic grading parameters Fusion Yes = 1 No = 0 Amount ofbone 5 80-100%  4 60-80% 3 40-60% 2 20-40% 1  0-20%

Posteroanterior radiographs of all animals were taken post-operatively,at 4 weeks, and at 12 weeks following harvest. Representative Xrays ofeach group are shown in FIGS. 4-9. Granules were evident inpost-operative Xrays for Groups 1, 2, 3, 4, 5 and 6. At the four-weektime point, few granules were visible for Group 1 and 2, though theirpresence could be noted. More granules were apparent in Group 3 and 4,while Group 6 clearly showed remaining particles or granules. At 12weeks, particles or granules could not be distinguished from bone forGroups 1, 2 and 3. Group 4 showed little bone formation and no clearlyvisible particles. Particles were still evident at 12 weeks for Group 6.

Radiographic assessment was performed by 3 blinded observers. Thegrading consisted of a binomial assessment of fusion and a 5-levelassessment of the amount of bone present within the fusion mass. Resultsare shown in Table 11. The mean value of individual grades for eachanimal were first calculated, then the mean and standard deviation foreach group was calculated.

TABLE 11 Pooled results of grading showing mean and (standard deviation)Group Left Fusion Right Fusion Left Bone Right Bone 1 0.777(0.17)  1(0)4.83(0.27) 4.11(0.68) 2 0.777(0.40) 0.833(0.40) 3.50(1.09) 4.22(0.75) 30.666(0.51) 0.833(0.40) 3.44(1.40) 3.06(1.25) 4 0.222(0.40) 0.166(0.40)1.11(0.17) 1(0) 5 0.277(0.44) 0.611(0.38) 3.06(1.28) 3.06(1.25) 6  1(0) 1(0) 4.78(0.17) 4.83(0.18)

Example 11: In Vivo Canine Segmental Ulnar Defect Study

Osteoinductive activity of a homodimeric protein (Hp) including arecombinant polypeptide produced according to Example 6 (i.e., SEQ IDNO: 260, including intramolecular C44-C48 and intermolecular C80-C112and C79-C114 disulfide bonds) and porous beta-tricalcium phosphate(β-TCP) as a carrier material was evaluated in canine segmental ulnardefect model. The calcium phosphate carrier (e.g., β-TCP) had a calciumto phosphate ratio of about 0.7 to about 1.5, and the porosity of β-TCPwas more than 70% with pore size from about 300 μm to about 600 μm.

Experimental Design

The protocol of this study was approved by the Nippon Veterinary andLife Science University Committee for Animal Experimentation. Animalexperiments were carried out in accordance with the National Institutesof Health guidelines for the care and use of laboratory animals. 2.5-cmcritical-size segmental ulnar defects were created in 15 forelegs of 8dogs. In all cases, the diameter of the resected ulna was 7-8 mm, andthe volume of the osteotomized ulna was approximately 0.96-1.26 cm³. Ateach defect site, 700 mg of artificial bone (β-TCP) was implanted withthe homodimeric protein. The amount of homodimeric protein (0, 35, 140,560, or 2240 μg) varied across each of 5 experimental groups (control,Hp 35, Hp 140, Hp 560, and Hp 2240, respectively). Each group consistedof 3 forelegs. Radiographic examination was performed every week afterthe operation, and computed tomography (CT) was performed every 4 weeks.

In alternative embodiments, at each defect site, 800 mg of artificialbone (β-TCP) was implanted with the homodimeric protein. The amount ofhomodimeric protein (160, 480, 640, or 1600 μg) varied across each of 4experimental groups (Hp 160, Hp 480, Hp 640, and Hp 1600, respectively).Each group consisted of 3 forelegs. Radio-graphic examination wasperformed every week after the operation, and computed tomography (CT)was performed every 4 weeks.

Preparation of Implant Materials

The implant materials were composed of homodimeric protein and β-TCP(HOYA Corp., Tokyo, Japan). Freeze-dried homodimeric protein powder wasreconstituted in sterilized distilled water (Otsuka Pharmaceutical Co.Ltd., Tokyo, Japan) before use. Before implantation, 700 mg of β-TCP(approximately 1.89 ml) was soaked for 15 min at room temperature in0.63 ml of distilled water containing 1 of the 4 doses of homodimericprotein. As a control, 700 mg of β-TCP soaked in 0.63 ml of plaindistilled water was used. The individual β-TCP granules were 2-4 mm indiameter, with an interconnecting porous structure (pore size 50-300 μm,porosity 75%). This granule size is the most convenient for manipulationand tight implantation.

The implant volume, size of β-TCP granules, and volume of homodimericprotein solution were selected based on the results of pilot studiesconducted prior to the experiment. Although the maximum implantationvolume of β-TCP granules was determined to be 800 mg, 700 mg of β-TCPwas used in this study due to inter-individual variability.Additionally, our pilot data indicated that 1.0 g of β-TCP granulescould soak up as much as 1.0 ml of distilled water. To avoid theaccumulation of residual fluid, 0.9 ml of homodimeric protein solutionwas used for per 1.0 g of β-TCP granules; thus, 700 mg of β-TCP granuleswas treated with 0.63 ml of the distilled water containing homodimericprotein.

Animal Model

All 8 dogs used in this study were healthy female 1-year old beagleswith a body weight of 9.5-11.3 kg. The dogs were anesthetized via anintravenous injection of propofol (7 mg/kg); post-intubation, theanesthesia was maintained with isoflurane (1.5-2.0%) in oxygen. Whilethe dogs were under general anesthesia, both their forearms wereprepared and draped in a sterile environment. The lateral skin wasincised longitudinally, and soft tissue was divided between the lateraldigital extensor muscle and the ulnaris lateralis muscle to expose theulna and the interosseous ligament. An oscillating saw was used tocreate the 2.5-cm segmental osteoperiosteal defects distally to theinterosseous ligament. The periosteum around the bone defects wasremoved completely, and then the β-TCP granules were implanted tightly.The longitudinal length of the defects was about 3 times theperpendicular length, and represented a critical-size defect that doesnot heal spontaneously. After the β-TCP granules were implanted, themuscles were sutured using 3-0 absorbable monofilament sutures. The skinwas closed using a 3-0 nylon monofilament suture. Perioperativeanalgesia was maintained by pre- and post-operative subcutaneousadministration of buprenorphine (0.02 mg/kg), which was administeredtwice a day for 3 days after surgery. For 7 days after surgery, 25 mg/kgampicillin was orally administered twice a day.

X-Ray and CT Examination

Lateral view X-rays of the dogs' forelegs were collected prior to,immediately after, and once a week for 12 weeks after the operation. Analuminum plate (25 mm×74 mm) was exposed with the forelegs tostandardize the magnification and contrast difference in each X-rayimage. The width of the regenerated bone was measured at the middlesection.

To measure the cross-sectional area and mineral density of theregenerated bone, CT imaging (Asteion; Toshiba Medical Systems Corp.,Tochigi, Japan) was performed immediately after, and at 4, 8, and 12weeks after surgery while the dogs were maintained under generalanesthesia. Multiplanar reconstruction (MPR) mode was used forexamination. Twenty-five 1-mm-thick slices were reconstructed in thedefect with correct alignment, and the thirteenth slice was defined asthe objective center slice for measurement. The window width and windowlevel were set at 1500 and 300 HU (Hounsfield units). Thecross-sectional area was measured by manually surrounding the outline ofthe regenerated bone. Quantitative CT (QCT) was used to measure bonemineral density in the transverse plane. QCT is a volumetric method thatestimates the milligrams of hydroxyapatite per cubic centimeter of bonetissue. Slice thickness was maintained at 1 mm, and a calibrationphantom was scanned with the forelegs. The mean CT number value (HU) inthe specified region of interest (ROI, diameter=5.5 mm) was determinedat the center of the regenerated bone and the calibration phantom(B-MAS200; Kyoto Kagaku Co. Ltd, Kyoto, Japan), after which the meanbone mineral density was calculated.

Results are presented as mean (SD). A two-way repeated-measures ANOVAwas used to investigate the effects of the different homodimericprotein-dose treatments; differences among the means were analyzed withTukey-Kramer's post hoc tests. Significance was defined as P<0.05.

Results

FIGS. 10a-y are radiographs showing the post-operative change in eachgroup. One week after surgery, only 2 cases (1 each from the Hp 2240 andHp 560 treatment groups) showed slight radiopaque callus lines aroundthe donor site. Two weeks after surgery, remarkable callus formation wasobserved in all cases from the Hp 2240 and Hp 560 treatment groups. Inthe former, the massive callus possessed irregular outlines and includedthe distal and proximal parts of the ulna (FIG. 10b ). In the latter,the radiopaque callus line was observed just around the donor site (FIG.10g ). One case from the Hp 140 group displayed slight radiopaque calluslines around the donor site. No callus formation was observed in the Hp35 and control groups (FIG. 10q, 10v ).

Four weeks after surgery, all cases in the Hp 2240 and Hp 560 treatmentgroups possessed radiodense, well-demarcated callus formation, and theborderline between the implant materials and the host bone was almostunclear; the granularity of the implanted materials had disappeared inall cases within both treatment groups (FIG. 10c, 10h ). The callusexpansion areas were larger among Hp 2240 cases than Hp 560 cases. Inthe Hp 140 group, there was minor callus formation around the implantedmaterials in 1 case only. However, the granularity of the implantedmaterials had disappeared in all cases (FIG. 10m ). No callus formationwas observed in the Hp 35 group, and the granularity of the implantedmaterials had disappeared in only 2 cases (FIG. 10r ). In all cases ofthe control group, no visible changes were observed in the center area,but a slight reduction of the mineral density was seen in the proximaland distal part of the grafted materials. Widening of the gap betweenthe proximal ulna and implanted materials was also observed in thesecases (FIG. 10w ).

Eight weeks after surgery, the remodeling process had progressed in theHp 2240 and Hp 560 groups, with the outline of the callus having changedto fit the shape of the host bone (FIG. 10d, 10i ). In the Hp 2240group, the borderline between the regenerated bone and the host bonedisappeared in 2 cases at the proximal side, and in all cases at thedistal side (FIG. 10d ). In the Hp 560 group, the borderline disappearedonly at the distal side in all cases (FIG. 10i ). Although 1 case in theHp 140 group displayed a disappearance of the borderline at the distalside, all others possessed well-demarcated gap lines at both sides ofthe bone (FIG. 10n ). There were no connections on either side betweenthe regenerated bone and the host bone in the Hp 35 group (FIG. 10s ).In the control group, the β-TCP granules had been resorbed and theradiolucent area had increased noticeably (FIG. 10x ).

Twelve weeks after surgery, all cases in the Hp 2240 group showed a muchlarger and wider bone regeneration (FIG. 10e ). The radiolucentborderline between the regenerated bone and the host bone disappeared atboth sides in 2 cases, but a slight borderline remained at the proximalside in 1 case. In all cases of the Hp 560 group, the borderlinedisappeared at the distal side (FIG. 10j ). A narrow borderline waspresent at the proximal side, but it was covered by callus. The width ofthe regenerated bone was larger than that of the proximal end of theulna (FIG. 10j ). In one case in the Hp 140 group, the borderlinedisappeared at the distal side, but remained at the proximal side; theborderlines were present at both the sides in the other 2 cases (FIG.10o ). The width of the regenerated bone was almost equal to or lessthan that of the proximal end of the ulna (FIG. 10o ). There were nocases in the Hp 35 group where the regenerated bone connected to thehost bone; visible radiolucent gap lines were present along both sidesof the bone in all cases (FIG. 10t ). The width of the regenerated bonewas much smaller than that of the proximal end of the ulna (FIG. 10t ).No bony tissue was regenerated at the donor sites in the control group,and the β-TCP granules had resorbed even further since the previousexamination (FIG. 10y ). Example 12: In Vivo Osteoinductive Activity inSheep Interbody Fusion Model

Osteoinductive activity of a homodimeric protein including recombinantpolypeptides produced according to Example 6 (i.e., SEQ ID NO: 260,including intramolecular C44-C48 and intermolecular C79-C112 andC80-C114 disulfide bonds), porous beta-tricalcium phosphate (β-TCP) as acarrier material and a peek cage as an accommodator was evaluated in aninterbody fusion model in sheep. The calcium phosphate carrier has acalcium to phosphate ratio of about 0.7 to about 1.7.

Pre-Surgery Preparation

Animals (Species: Ovis Aries; Breed: Border Leicester Merino Cross;Source: UNSW approved supplier—Hay Field Station, Hay, NSW and animalswere purchased following UNSW Animal Care and Ethics Committee approval;Age: 4 years old age; and Gender: Female (Ewe)) were prepared forsurgery according to standard operating procedures. Twenty-four hoursprior to surgery, pre-emptive analgesia was administered, by applying atransdermal fentanyl patch (100 mg-2 mcg/kg/hr) to the right foreleg ofeach animal (left foreleg was used for iv line). Prior to application,the wool was clipped and the skin cleaned with alcohol swabs to ensureadequate absorption. Animals were fasted and water withheld a minimum of12 hours prior to surgery.

Surgery

Sheep allocated to the study were randomly selected on the day ofsurgery. Once a sheep was selected it was assigned a number andear-tagged according to standard operating procedures. Thisidentification number was recorded in the study notebook.

On the day of surgery and prior to commencement, the Study Veterinarianexamined each animal to ensure that it was free of disease or anycondition that might interfere with the purpose or conduct of the study.Notes were made in the Study Notebook against the animal number as tothe condition of each animal and its suitability for inclusion in thestudy.

Animals were induced, anaesthetised, maintained, and monitored duringthe procedure according to standard operating procedures. The leftforeleg was used for cephalic intravenous access. Blood was taken forpre-operative analysis according to standard operating procedures, priorto i.v. administration of Hartman's solution. Blood samples werelabelled “PRE-OP” along with study ID, animal number and date and sentto IDEXX Australia for routine biochemistry (4 ml) and haematology (4ml).

Surgery was performed according to a modified version of standardoperating procedures.

Surgical Procedure—L45 XLIF+Pedicle Screws

Prior to surgery, all animals were under food restriction (NPO) forforty-eight hours and housed in the isolation pen care facility.Following administration of anesthetic medications and induction ofgeneral anesthesia, the posterior lumbar region, iliac crest andproximal tibia were aseptically prepared.

Graft Mixing Procedure

The homodimeric protein (10 mg/vial) was dissolved in 0.3 mL distilledwater to make a 33.3 mg/mL HP stock solution A. A block of β-TCP(approx. 150 mg) was placed into an interbody cage. For each group, “Hpsolution” that was diluted or divided from HP stock solution A wasdropped evenly across the block of β-TCP (approx.150 mg) placed on asterilized Petri dish. After the fluids were completely dropped, theblock of β-TCP was allowed to stand for more than 15 minutes at roomtemperature before implantation.

Group A-F used the above-described homodimeric protein (Hp) combinedwith β-TCP carrier as graft material, with the homodimeric protein doseper site shown in Table 12. Preparations of mixing procedures were asfollows.

TABLE 12 Carrier Hp Pedicle Time Group Animal βTCP mg/site Level Cagescrews point A 1 150 mg 4.0 mg L45 Yes Yes 12 wks B 2 150 mg 2.0 mg L45Yes Yes 12 wks C 3 150 mg 1.0 mg L45 Yes Yes 12 wks D 4 150 mg 0.5 mgL45 Yes Yes 12 wks E 5 150 mg 0.1 mg L45 Yes Yes 12 wks F 6 150 mg   0mg L45 Yes Yes 12 wks G 7 150 mg Autograft L45 Yes Yes 12 wks (iliaccrest) A 8 150 mg 4.0 mg L45 Yes Yes 12 wks B 9 150 mg 2.0 mg L45 YesYes 12 wks C 10 150 mg 1.0 mg L45 Yes Yes 12 wks D 11 150 mg 0.5 mg L45Yes Yes 12 wks E 12 150 mg 0.1 mg L45 Yes Yes 12 wks F 13 150 mg   0 mgL45 Yes Yes 12 wks G 14 150 mg Autograft L45 Yes Yes 12 wks (iliaccrest) X 15 150 mg   0 mg L45 Yes Yes  0 wks

For group A, Hp 4 mg/site: Prepared Hp stock solution A (33.3 mg/mL) byadding 0.3 mL injection water to each of the glass vials with 10 mghomodimeric protein in it as Hp stock solution A. Put a β-TCP block(approx. 150 mg) into the peek cage. Delivered 120 μL Hp stock solutionA by drops evenly into 150 mg β-TCP block.

For group B, homodimeric protein 2 mg/site: Mixed water and Hp stocksolution A in a 1:1 volume:volume ratio, then obtained Hp solution B(16.7 mg/mL). Put a β-TCP block (approx. 150 mg) into the cage.Delivered 120 μL Hp solution B by drops evenly into 150 mg β-TCP block.

For group C, homodimeric protein 1 mg/site: Mixed water and Hp stocksolution B in a 1:1 volume:volume ratio, then obtained Hp solution C(8.3 mg/mL). Put a β-TCP block (approx. 150 mg) into the cage. Delivered120 μL Hp solution C by drops evenly into 150 mg β-TCP block.

For group D, homodimeric protein 0.5 mg/site: Mixed water and Hp stocksolution C in a 1:1 volume:volume ratio, then obtained Hp solution D(4.2 mg/mL). Put a β-TCP block (approx. 150 mg) into cage. Delivered 120μL Hp solution D by drops evenly into 150 mg β-TCP block.

For group E, homodimeric protein 0.1 mg/site: Mixed water and Hp stocksolution D in a 1:4 volume:volume ratio, then obtained Hp solution E(0.8 mg/mL). Put a β-TCP block (approx. 150 mg) into cage. Delivered 120μL Hp solution E by drops evenly into 150 mg β-TCP block.

For group F, homodimeric protein 0 mg/site: Put a β-TCP block (approx.150 mg) into cage. Delivered 120 μL water by drops evenly into 150 mgβ-TCP block.

Interbody Peek Cage

The transverse processes were palpated to identify the appropriatespinal levels. The level was verified by fluoroscopy. Caspar pins wereplaced into the L4 and L5 vertebral bodies and a Caspar retractor usedto distract the disc space. The disc was removed with sharp dissectionand curettes and the endplates prepared. The interbody device filledwith the graft material was carefully placed into the disc space and theretractors released. The soft tissues were re-apposed and the skinclosed in layers.

FIG. 11 shows the appearance of an interbody cage.

Pedicle Screws

Following completion of the XLIF, the animal was repositioned in theprone position and draped using sterile technique; an initial skinincision was made in the dorsal mid-line of the low back centered overthe L3-S1 levels. Blunt dissection using a Cobb elevator andelectrocautery, when necessary, was performed in the sagittal planealong the neural arch—permitting exposure of the L45 facets andtransverse processes and insertion of pedicle screws and rods at thislevel.

Radiographs were taken immediately following surgery in thepostero-anterior plane using a mobile x-ray machine (POSKOM) and digitalcassettes (AGFA). The data were stored in DICOM format and exported toJPG images using ezDICOM medical viewer software. This is performedaccording to standard operating procedures.

Post-Operative Monitoring

Animals were monitored daily for the first 7 days and recorded. Animalswere examined at least once daily for the duration of the study byveterinary technicians and recorded weekly. Any health concernsidentified by the technicians were reported to the veterinary staff forfurther evaluation and management by the PI.

Sheep were monitored as per the study site standard operating procedure.The surgical incision, appetite, changes in skin and hair, eyes andmucous membranes, respiratory system, circulatory system, posture/gait,behaviour patterns (occurrence of tremors, convulsions, excesssalivation, and lethargy) were monitored daily for the firstpost-operative week and weekly thereafter. Signs monitored thereafterwere alertness/attentiveness, appetite, and surgical site, appearance ofeyes, ambulation, and ability to keep the head raised. Criteria forintervention were signs of infection.

Post-operatively, animals received oral antibiotics (Kelfex) andanalgesics (buprenorphine, 0.005-0.01 mg/kg IM) for the first 3 days.Daily neurological assessments were made for the first 7 dayspost-operatively. Post-operative pain relief was provided thereafterbased on clinical monitoring.

Explant

At the designated time point, each animal had its identification numberconfirmed before being induced and anaesthetized, as per standardoperating procedures.

After anaesthetic induction, blood was taken from either the jugular orcephalic vein, according to standard operating procedures. Samples werelabelled “with study ID, animal number and date. The samples weretransported to SORL in a sealed biohazard bag and maintained below 30degrees C. and sent to IDEXX Australia for Routine biochemistry (4 mLs)and haematology (4 mLs). Transportation times were noted in thenotebook.

While still under an anesthesia, animals were euthanized by lethalinjection of Lethobarb according to standard operating procedures. Thecarcass was transported immediately to SORL and kept at a temperature ofless than 30 degrees C.

Each animal was examined and dissected according to standard operatingprocedures. The Lumbar Spine was harvested and photographed using adigital camera. The surgical sites were examined for signs of adversereaction or infection and the results noted and photographed.

Immediately after harvest, the stability of the fusion mass was assessedby manual palpation in all animals 12 weeks. Two trained and experiencedblinded observers worked together to assess the fusion mass in lateralbending and flexion-extension with the pedicle rods intact as well asremoved.

The fusions were graded as either fused (rigid, no movement) or notfused (not rigid, movement detected) when manual palpation evaluated inlateral bending on the right and left sides and flexion-extension at thetreated level. The mobility of the untreated level was used as arelative comparison at the time of manual palpation when evaluated inlateral bending on the right and left sides as well asflexion-extension.

Range of Motion (ROM) Testing

The L45 segments were carefully harvested from the spine. A 4 mm×15 mmscrew was inserted into the vertebral bodies and used to assist inpotting the samples.

The segments were carefully potted in a resin for ROM evaluation. Rangeof motion in flexion-extension (FE), lateral bending (LB) and axialrotation (AR) were determined using a Denso Robot. The rods were removedprior to testing. A 7.5 Nm pure moment will applied to the spines in FE,LB and AR and resulting angular deformation recorded with the testingequipment. Each loading profile were repeated 3 times and a mean valuefor FE, LB and AR obtained for each treated level as shown in FIG. 12.Samples were fixed in phosphate buffered formalin after mechanicaltesting for paraffin histology on one side and PMMA histology for theother side of the fusion as outlined below. ROM data were analysed usingANOVA using SPSS.

As shown in Table 13, manual palpation indicated non-rigid motionsegments for doses below 0.5 mg, and rigid motion segments for doses of0.5 mg and above, as well as for autograft.

TABLE 13 Manual Manual Time Palpation Palpation Group Animal ID Hpmg/site (weeks) FE* LB{circumflex over ( )} X 15 W2780   0 mg 0 notrigid not rigid F 6 W2781   0 mg 12 not rigid not rigid F 13 W2792   0mg 12 not rigid not rigid E 5 w2790 0.1 mg 12 not rigid not rigid E 12W2791 0.1 mg 12 not rigid not rigid D 4 W2788 0.5 mg 12 rigid rigid D 11W2789 0.5 mg 12 rigid rigid C 3 W2786 1.0 mg 12 rigid rigid C 10 W27871.0 mg 12 rigid rigid B 2 W2784 2.0 mg 12 rigid rigid B 9 W2785 2.0 mg12 rigid rigid A 1 W2782 4.0 mg 12 rigid rigid A 8 W2783 4.0 mg 12 rigidrigid G 7 W2793 Autograft 12 rigid rigid (Iliac Crest) G 14 W2794Autograft 12 rigid rigid (Iliac Crest) *FE: Flexion extension{circumflex over ( )}LB: Lateral bending

As shown in FIG. 12, range of motion showed little change for anytreatment in axial rotation. However, flexion extension decreased fromintact for all treatment groups and showed a trend towards increasedstability with increasing homodimeric protein dose. Autograft and 1.0 mghomodimeric protein were the most comparable. Lateral bending wasreduced for all treatments as well, to <50% of the intact value for alltreatments. Lateral bending also showed a reduction in ROM as withincreasing homodimeric protein dose. The dose influence was mostprevalent at the 0.1 mg to 0.5 mg stage.

Micro Computed Tomography—Spine

Micro Computed tomography (μCT) was performed on the spines using anInveon Scanner (Siemens, USA). Slice thickness were set to approximately50 microns for all scans. CT scans were stored in DICOM format. Threedimensional models were reconstructed based and examined in the axial,sagittal, and coronal planes. The DICOM stacks were sent to the StudySponsor for additional analysis.

Axial, sagittal and coronal images as well as anterior and posterior 3Dmodels were provided for each animal. The micro-CT reconstructions wereevaluated by reviewing the coronal and sagittal planes to examinefusions between the treated levels. The micro-CT were graded using thesame Radiographic grading score (Table 14) considering the entiremicro-CT stack by two trained and experienced observers blinded totreatment groups. Each fusion will also be graded with a scale of 1 to 4representing the amount of bone qualitatively at the level: 0-25%, 2:26-50%, 3: 51-75%, 4: 76-100%.

TABLE 14 Grading scale for micro CT Number Grade Description 0 No newbone No new bone formation visible 1 Visible new bone New bone formationvisible but no continuous bone 2 Visible new bone Continuous bridgingnew bone with visible lucency 3 Probable fusion Continuous bridging newbone formation

Representative images of the micro-CT for each animal were prepared inthree orthogonal planes as well as three dimensional models in theanterior and posterior views. Micro computed tomography (μCT) scanningwere performed on all animals following radiography using a SiemensInveon in-vivo microcomputer tomography scanner to obtain highresolution radiographic images of the spinal fusions in three planes.This was performed according to standard operating procedures; however,in addition thicker reconstructions were also be taken and examined foreach animal using a 500 micron summation image technique. Note, the 3Dreconstructions were reviewed to evaluate the overall fusion status andrepresentative images were provided in the report and appendices for allanimals. This was performed according to standard operating procedures.The sagittal and coronal CT images were reviewed and an overall gradefor the fusion given based on Table 14. See FIGS. 13-15.

Fusion Grading

0 Hp mg/sites demonstrated residual TCP and some bone formationprimarily at the endplate. With 0.1 Hp mg/site new bone was generatedand minimal residual TCP was present but solid bone bridging was notpresent. With 0.5 Hp mg/site good bone quality was generated but withthe presence of some lucent lines within the graft. With doses of 1.0 Hpmg/site and 2.0 Hp mg/site the inter-cage space was filled with goodquality bone with minimal lucent areas. 4.0 Hp mg/site generated a highgrade of bone based on volume; however, there was lucency within thebone including some large and small pockets. Autograft (iliac crest)demonstrated variable results with fair to good bone formation and areasof non-union. The following table summarizes the grading of each site.

Fusion grading based on Micro CT analysis were as shown in Table 15.Overall bone grade and fusion grade peaked with 1.0 and 2.0 mg Hp doses.Bone grade was graded with a scale of 1 to 4 representing the amount ofbone qualitatively at the level: 0-25%, 2: 26-50%, 3: 51-75%, 4:76-100%. Fusion grading was done from 0-3 based on 0-No new bone,1-visible new bone, but not continuous, 2-possible fusion with lucency,3-probable fusion with bridging bone.

TABLE 15 Group ID Hp mg/site Time Bone Fusion F W2781 0 mg 12 2 1 FW2792 0 mg 12 2 1 E w2790 0.1 mg 12 3 1 E W2791 0.1 mg 12 3 1 D W27880.5 mg 12 4 2 D W2789 0.5 mg 12 4 2 C W2786 1.0 mg 12 4 2 C W2787 1.0 mg12 4 3 B W2784 2.0 mg 12 4 2 B W2785 2.0 mg 12 4 3 A W2782 4.0 mg 12 4 2A W2783 4.0 mg 12 3 2 G W2793 Autograft(Iliac Crest) 12 4 2 G W2794Autograft(Iliac Crest) 12 2 2

Example 13: Controlled Release System Preparation (Double EmulsionMethod/Basic Substance/Hydrophilic Drug)

In one embodiment, 0.25 g of PLGA (Lactic acid/Glycolic acid ratio65/35, MW 40000-75000, Sigma-Aldrich) dissolved in 2.5 mL ofdichloromethane (Merck) was shaken with a shaker (1000 rpm) for 5minutes to form a 10% PLGA solution (10% oil phase solution). 2.5 mLdouble-distilled water (DDW) was slowly mixed the 10% PLGA solution andstirred at 1,000 rpm for 15 minutes to form a first emulsion (w/o). Thefirst emulsion was added to 10 mL of a 0.1% (w/v) polyvinyl alcohol(PVA) (MW˜13000, Fluka) second aqueous solution and stirred at 500 rpmand evacuated the gas for 5 minutes to form a second emulsion (w/o/w).The second emulsion was continuously stirred for 4 hours and then leftstanding for one minute. Particles in the pellet were collected bycentrifugation at 4,000 rpm for 5 minutes. The particles were washedwith 5 mL of DDW for minutes. After centrifugation and washing threetimes, centrifuged particles were collected and lyophilized 3 days toform PLGA microparticles. 2 mg and/or 4 mg β-TCP powder (Sigma-Aldrich)was mixed with 504 DDW and 10 μg of the homodimeric protein (Hp)including recombinant polypeptide produced according to Example 6 (i.e.,SEQ ID NO: 260) to form a slurry. The slurry was then mixed or coated onthe surface of 50 mg PLGA microparticles and lyophilized 3 days to formPLGA microparticles, one of the controlled release system. In certainembodiments, the lyophilized controlled release system could be pressedto form a flat piece.

In alternative embodiment, 2.5 mL dichloromethane was mixed with 0.25 gof poly lactic-co-glycolic acid, PLGA 65:35 (Supplier, Sigma) andstirred (1000 rpm) for 5 minutes which became a 10% oil phase solution(P1). 0.25 mL of double-distilled water (DDW) was added into P1 andstirred (1000 rpm) for 15 minutes which became first emulsion phase(w/o, P2). The P2 was placed into 10 mL of 0.1% (w/v) polyvinyl alcohol(PVA) (MW˜13000, Fluka) and stirred (500 rpm) for 4 hours (P3).Centrifugation of P3 at 4,000 rpm for 5 minutes, the supernatant wasdiscarded, and the residual solution was collected. 5 mL of PBS wasadded into P3 and repeat for three times, the residual solution wascollected and lyophilized. The lyophilized powder of PLGA microspherewas weighted, and the production rate (%) was calculated. The 0.06 mL ofDDW was mixed with 2 mg of β-TCP powder (Sigma-Aldrich), then 20 μg ofthe homodimeric protein was added into β-TCP for stirring 5 minutes.After that, 50 mg of PLGA microsphere was added into the mixture andstirred evenly. The microsphere containing was lyophilized and pressed atablet with a size Φ10 mm. (The appropriate pressure was 5˜10 Kg).

In some embodiments, the 2 mg and/or 4 mg β-TCP powder could be replacedwith either 4 mg tricalcium phosphate (TCP) or 1 mg alpha-tricalciumphosphate (α-TCP). In certain embodiments, the PLGA65/35 could bereplaced with PLGA50/50, polylactic acid (PLA) or polyglycolic acid(PGA).

Evaluation of Homodimeric Protein (Hp) Release from PLGA/Hp-β-TCP

100 mg of PLGA/Hp-β-TCP was soaked in 1 mL human serum, and shaken with60 rpm at 37° C. Human serum solution containing released homodimericprotein was collected at 15 min, 1 hour, day 1, 2, 3, 7, 10 and 14, andat each time point was replaced with 800 μL of fresh human serum. Thecollected human serum was stored at −80° C. and all samples wereanalyzed simultaneously with a direct ELISA assay.

The release profile of the PLGA microparticles coated with β-TCP and thehomodimeric protein is shown in FIGS. 16a and 16b . Homodimeric proteinphysically adsorbed on the surface of the PLGA microparticles was shownto continuously release therefrom to an in-vitro solution via diffusionand PLGA hydrolysis. Results from an ELISA kit showed that 17% and 31.5%of relative amount of homodimeric protein was released at 15 min and 1hr, respectively. The relative releasing percentages of homodimericprotein were 14.5% (at 60 min to day 1), 14.3% (day 1 to day 2), 7.6%(day 2 to day 3), 9.3% (day 3 to day 7), 5.4% (day 7 to day 10) and 0.4%(day 10 to day 14) and that has shown a slow-release pattern. Thisformulation of PLGA/Hp-β-TCP alleviated the common issue of burstrelease [Giteau et al., Int J Pharm 350:14 (2008)]; most deliverysystems deliver a burst release during the first few hours, oftenreleasing over 60% of the encapsulated/surface bound product [Woodruffet al., J Mol Histol 38:425 (2007) and Sawyer et al., Biomaterials30:2479 (2009)].

FIG. 17 shows morphology and a diameter distribution of the PLGAmicroparticles under an electron microscope. The PLGA microparticleswere globular and had a diameter distribution ranging from 100 μm to 150μm.

In another embodiment, 2 g of PLGA was dissolved in 20 mL ofdichloromethane (DCM) to form a 10% PLGA/DCM solution. Biphasic calciumphosphate (BCP) powder was dispersed in water to form an aqueoussolution. The aqueous solution was then mixed with the PLGA/DCM solutionand stirred with a magnetic stirrer for 30 minutes to form an emulsion.Next, the emulsion was fed into a granular machine to perform a spraygranulation process to form PLGA microparticles.

Evaluation the New Bone Formation of PLGA/Hp-β-TCP on Balb/C MiceOsteonecrosis Model

Surgery Procedure

In the animal osteonecrosis model, in order to simulate the realosteonecrosis situation, the whole tibia periosteum was stripped. A 2 mmlength of the mid-shaft of the tibia on the right side of a mouse wascut out with a saw. The cut surface of the bone was frozen using liquidnitrogen for 5 min to mimic necrotic bone. Next, the fragment wasreversed and put back to its original site in the tibia and fixed toboth ends with the other parts of the tibia by using a syringe needle(No. 26) as an intramedullary fixation. After a test article was placedaround the bone fracture, the wound was closed with silk sutures. Themice were divided into six groups, including necrotic bone control (C),PLGA/β-TCP (PT), PLGA/0.2 μg Hp-β-TCP (POT-0.2), PLGA/0.8 μg Hp-n-TCP(POT-0.8), PLGA/1.6 μg Hp-3-TCP (POT-1.6) and PLGA/3.2 μg Hp-β-TCP(POT-3.2) groups. Three to six mice in each experimental group wereobserved at 4 weeks after surgery.

Soft X-Ray Observation

At 4 weeks after the operation, the tibia bone fractures wereradiographically examined by soft X-rays (SOFTEX, Model M-100, Japan) at43 KVP and 2 mA for 1.5 s. The appropriate magnification was appliedthroughout the observation period, and the resultant micrographs werecompared among all carriers together with controls.

FIG. 18 shows the X-ray photographs of mice tibia osteonecrosis fragmentcallus formation at 4 weeks after implantation of PLGA/Hp-β-TCPcontaining different homodimeric protein doses compared to control (C)or PLGA/β-TCP (PT) groups. An incomplete fusion was noted in controlgroup, and a small gap was existed in osteonecrosis region in PT group.Clear fusion masses were observed in POT-0.2, POT-0.8, POT-1.6 andPOT-3.2 groups. The results indicated that the efficacy of bone repairon PLGA/Hp-β-TCP groups were greater than control and PT groups.

Histological Analysis of Bone Tissue

Histochemical analyses were concurrently employed to assess themicroscopic changes in the bone tissue. Prior to hematoxylin-eosin (H&E)staining, all samples of bone tissue were decalcified using 0.5% EDTA.The resultant samples were embedded into paraffin wax, and 5 μm sectionswere prepared. Sections were routinely stained with H&E and observedwith a microscope. At 400× magnification, the callus area was comparedwith that of the control group.

As shown in FIG. 19, the new bone formation was evaluated after 4 weeksof implantation of PLGA/Hp-β-TCP (POT). The bone formation rate of thePLGA/β-TCP (PT) group show a similar result as compared to the controlgroup. Bone formation rate was enhanced in the POT groups as compared tothe PT and control groups, which was increased in a dose-dependentmanner except for the POT-3.2 group. These results demonstratedpotential advantages of homodimeric protein controlled-release carriersthat can induce bone regeneration on Balb/C mice osteonecrosis model.

Example 14: Sustained Release System

Putty Preparation

Powders were prepared and mixed according to the formulas in Table 16.The powders were stored at 4° C. overnight. On the day that the puttywas prepared, all materials (i.e., the powder, β-TCP, glycerol anddeionized water) were illuminated with UV light for 20 minutes.According to the animal experimental bone defect range of 2×0.5×0.5 cm,putty weights of about 0.9 g were prepared in accordance with theformulas as shown in Table 16.

TABLE 16 Putty formulation Formula A Formula B Formula C Formula DFormula E Formula F Formula G Powder CaSO₄ · 1/2H₂O ^(*1) — 96% 96% 96%96% — — 0.864 g 0.864 g 0.864 g 0.864 g CaSO₄ · 2H₂O^(*2) — — — — — 48%48% 0.432 g 0.432 g HPMC^(*3) — 4% 4% 4% 4% 4% 4% 0.036 g 0.036 g 0.036g 0.036 g 0.036 g 0.036 g Ca₃(PO₄)₂ ^(*5) — — — — — 48% 48% 0.432 g0.432 g Liquid Glycerol^(*7) — 126 μl 126 μl 126 μl 126 μl 252 μl 252 μlHp^(*4) — — 40 μl of 40 μl of — 40 μl of 40 μl of 0.5 mg/ml 0.25 mg/ml0.25 mg/ml 0.5 mg/ml Deionized water — 54 μl 14 μl 14 μl 54 μl 68 μl 108μl L/P (Liquid/powder) — 0.2 0.2 0.2 0.2 0.4 0.4 β-TCP^(*6) (2-3 mm) 50mg 50 mg — 50 mg 50 mg 50 mg — Hp^(*4) 160 μl of 40 μl of — 40 μl of 40μl of 40 μl of — 0.125 mg/ml 0.5 mg/ml 0.25 mg/ml 0.5 mg/ml 0.25 mg/mlCross-section See Figure See Figure See Figure See Figure See Figure SeeFigure See Figure 20A 20B 20C 20D 20D 20D 20C ^(*1) Calcium SulfateHemihydrate (MT3, Taiwan), ^(*2)Calcium Sulfate Dihydrate (J.T. baker,USA), ^(*3)Hydroxypropyl Methylcellulose (Sigma-Aldrich, USA),^(*4)Homodimeric protein (Hp) including recombinant polypeptide producedaccording to Example 6 (i.e., SEQ ID NO: 260) final volume 20 μg,^(*5)Calcium Phosphate (Sigma-Aldrich, USA), ^(*6)Tricalcium PhosphateBeta Form (Wiltrom, Taiwan), ^(*7)Glycerol (Showa, Japan) Formula A: 160microliters of 0.125 mg/mL Hp solution was dripped in approx. 50 mgβ-TCP, in sterile conditions, and allowed to adsorb for 15 minutes.Formula B: 40 microliters of 0.5 mg/mL Hp solution was dripped inapprox. 50 mg β-TCP, in sterile conditions, and allowed to adsorb for 15minutes. Powder and liquid (as shown in Table 16 for Formula B) andpreviously prepared β-TCP granules were mixed together and molded.Formula C: Powder and liquid (as shown in Table 16 for Formula C) weremixed evenly. Formula D: 40 microliters of 0.25 mg/mL Hp solution wasdripped in approx. 50 mg β-TCP, in sterile conditions, and allowed toadsorb for 15 minutes to form Hp/β-TCP granules. Powder and liquid (asshown in Table 16 for Formula D) were mixed together to form a matrixand the matrix was molded in a specific shape. Hp/β-TCP granules wereevenly distributed in the outer layer of the matrix. Formula E: 40microliters of 0.5 mg/mL Hp solution was dripped in approx. 50 mg β-TCP,in sterile conditions, and allowed to adsorb for 15 minutes to formHp/β-TCP granules. Powder and liquid (as shown in Table 16 for FormulaE) were mixed together to form a matrix and the matrix was molded in aspecific shape. Hp/β-TCP granules were evenly distributed in the outerlayer of the matrix. Formula F: 40 microliters of 0.25 mg/mL Hp solutionwas dripped in approx. 50 mg β-TCP, in sterile conditions, and allowedto adsorb for 15 minutes to form Hp/β-TCP granules. Powder and liquid(as shown in Table 16 for Formula F) were mixed together to form amatrix and the matrix was molded in a specific shape. Hp/β-TCP granuleswere evenly distributed in the outer layer of the matrix. Formula G:Powder and liquid (as shown in Table 16 for Formula G) were mixedevenly.

Sample Preparation

Formula putty shown in Table 16 were placed in 15 ml tube. Putty with orwithout β-TCP soaked with Hp was placed in 3 mL human serum, and allowedto stand at 37° C., under 5% CO₂. Human serum solution containingreleased Hp was collected at initial, 1 hour, Day 1, 2, 3, 7, 10, 14 and21, and at each time point was replaced with 2500 μL of fresh humanserum. The collected human serum was stored at −80° C. and all sampleswere analyzed simultaneously with a direct ELISA assay within the day.

OIF Quantification

To quantify the total concentration of homodimeric protein, an in vitrorelease test was used. Homodimeric protein concentrations in the humanserum were quantified using ELISA-methods (the assay was obtained frominVentive Health clinical systems, USA). The analyses were performedaccording to the instructions of the manufacturer. Briefly, samples, QCsamples and standards were added to 107 capture antibody (generated fromPharma Foods International Co., Ltd.) coated 96 well plates. Afterincubation and removal of the unbound substances, HRP-I07detectionantibody was added. This step was followed by a further washing step andincubation with a substrate. The color reaction was stopped and theoptical density measured at the appropriate wavelength. Theconcentration of homodimeric protein was back calculated off of thenon-linear regression of the standard deviations.

The purpose was to evaluate the release of homodimeric protein from abioresorbable osteoconductive composite such as beta-TCP or putty, andto assess its suitability for bone regeneration. Homodimeric proteinreleased from formula A was observed to have a burst release profile atthe beginning, but after the burst period (around 0 to 1 hour) aslow-release pattern as shown in FIG. 21 was observed. Compared withformula A, the homodimeric protein in formula B and C was wrapped up byputty or the matrix, so that it cannot be released at the beginninghours. In contrast, when homodimeric protein contained in β-TCP granulescovered distributed on the surface of the putty or the matrix, such asformula D, a sustained release effect was achieved. It was known thatputty was a bone graft substitute with a proven ability to acceleratebone regeneration. The composition of the putty determines plasticizingcapacity, hardening or curing. Thereafter, different proportions of theformulation, for example, the choice of calcium sulfate dihydrate orcalcium sulfate to prepare putty could achieve sustained release of thedosage form.

The development of bone substitute materials trends toward materialsthat are bio-absorbable, osteoconduction, osteoinduction, as well asbiocompatible. In other words, the direction of development forcomposite bone defect filling material is a material that ismulti-functional. In the designed putty, pores can be generated in thebone substitute for ingrowth of bone cells, while homodimeric proteincan be released over a long term for induction of osteoclasts andactivation of osteocytes in the slow process of material decomposition.Therefore, the healing of bone defects will be effectively accelerated.

Example 15: Clinical Study Design

Study Design 1

A Randomized, evaluator-blind, controlled study investigating theefficacy and safety of three dosage levels of the homodimeric protein(Hp) including recombinant polypeptide produced according to Example 6(i.e., SEQ ID NO: 260)/β-TCP in treatment of open tibial fractures withneed of bone grafting will be performed. A total of approx. 35 patientshaving initial open tibial fractures (Gustilo type IIIA or IIIB) willparticipate in the study and will be divided (randomized) into fourgroups and one control group (approx. 5 patients), each of the othergroups consisting of approx. 10 patients (vide infra). A vial contains5.5 mg lyophilized power of homodimeric protein. After reconstitution(the exact volume of water used to get the intended concentration willbe stated as in Table 17), the reconstituted homodimeric protein will bemixed with the β-TCP to make the final concentration of 1.5 mg/g (Group2), 2 m g/g (Group 3) or 3 mg/g (Group 4) the Hp/β-TCP, then certainamount of these mixture will be applied to the fracture site within 3months after the fracture occurred. Patients in the control group(Group 1) will receive autogenous bone graft but lacking the homodimericprotein and/or β-TCP. Subjects will be followed for efficacy and safetyfor the main study period of 30 weeks and an extension safety follow-upto 52 weeks after definitive treatment. In some embodiments, the totalamount of β-TCP used is based on the physician's judgment andadjustment.

TABLE 17 Hp Hp WFI Concentration Hp Volume β-TCP Final ConcentrationApplied (mg/vial) (mL) (mg/mL) required (mL) required (g) (Hp (mg)/β-TCP(g)) 5.5 3.0 1.8 2 2.4 1.5 (Group 2) 5.5 2.3 2.4 2 2.4 2.0 (Group 3)*5.5 × 2 1.5 × 2 3.6 1 × 2 2.4 3.0 (Group 4) [Two vials (1.5 ml for each(1 ml from each of Hp) Hp vial) vial, total 2 ml) *For finalconcentration 3.0 mg/g (Hp/β-TCP), 2 vials of lyophilized powder will bemixed with 1 vial β-TCP; each vial of lyophilized powder will bereconstituted by 1.5 ml WFI; and 1 ml from each reconstituted Hp (total2 ml) will be mixed with 1 vial (2.4 g) β-TCP.Patient Inclusion/Exclusion Criteria

Subjects will be included if ALL of the following Inclusion Criteriaapply:

The subject is ≥20 years old;

Females of non-childbearing potential or who have a negative result onpregnancy test within 72 hours prior to surgery, or males;

Initial open tibial fractures (Gustilo type IIIA or IIIB) and bone graftwithin 3 months of fracture;

In bilateral open tibial fractures, the random treatment assignment isfor the right tibia;

Definite therapy is performed within 3 months after the initial injury;and

Female subjects of childbearing potential (i.e., women who have not beensurgically sterilized or have not been post-menopausal for at least 1year) and male subject's partners of childbearing potential must agreeto use medically acceptable contraception methods throughout the studyperiod. Medically acceptable contraception methods include hormonalpatch, implant or injection intrauterine device, or double barriermethod (condom with foam or vaginal spermicidal suppository, diaphragmwith spermicidal). Complete abstinence can be considered an acceptablecontraception method. Oral contraceptive is an acceptable contraceptionmethod prior to the study, but an alternative method will be requiredduring the study;

Subjects will be excluded if ANY of the following Exclusion Criteriaapply:

Head injury with initial loss conscious;

Purulent drainage from the fracture, or evidence of activeosteomyelitis;

Compartment syndrome;

Pathological fractures; history of Paget's disease or otherosteodystrophy; or history of heterotopic ossification;

Endocrine or metabolic disorder that affects osteogenesis (e.g., hypo-or hyper-thyroidism or parathyroidism, renal osteodystrophy,Ehlers-Danlos syndrome, or osteogenesis imperfecta)

Has abnormal renal and/or hepatic functions, with Creatinine or ALTvalue>5 times the upper normal limit;

History of malignancy, radiotherapy, or chemotherapy for any malignancywithin the last 5 years;

An autoimmune disease (e.g. Systemic Lupus Erythematosus ordermatomyositis);

Previous exposure to rhBMP-2;

Hypersensitivity to protein pharmaceuticals, e.g, monoclonal antibodies,gamma globulins, and tricalcium phosphate;

Treatment with any investigational therapy within 28 days ofimplantation surgery;

Treatment for 7 days or more with prednisone (cumulative dose>150 mgwithin 6 months or other steroids with equivalent dose, refer toAppendix 1), calcitonin (within 6 months). Treatment of Bisphosphonates(for 30 days or more within 12 months), therapeutic doses of fluoride(for 30 days within 12 months);

The female subject who is lactating; and

Any condition that is not suitable to participate in the study based onthe physician's judgement.

Assessments of Efficacy

Primary Endpoint:

The primary study efficacy endpoint is the proportion of subjects whoreceived secondary intervention within 30 weeks after definitive woundclosure.

Secondary Endpoints:

The proportion of subjects who received secondary intervention withinpostoperative Week 6, Week 12, Week 18, Week 24, Week 42 and Week 52after definitive wound closure;

Time from definitive wound closure to secondary intervention;

Rate of clinical fracture healing within postoperative Week 6, Week 12,Week 18, Week 24, Week 30, Week 42 and Week 52 after definitive woundclosure;

Time from definitive wound closure to clinical fracture healing;

Rate of radiographic healing within postoperative Week 6, Week 12, Week18, Week 24, Week 30, Week 42 and Week 52 after definitive woundclosure;

Time from definitive wound closure to radiographic healing;

The term “secondary intervention” is in relation to any procedure thatis performed or any occurrence of an event that has the potential tostimulate fracture healing, including but not limited to bone graft,exchange nailing, plate fixation, nail dynamization, ultrasound,electrical stimulation, or magnetic field stimulation or others thatmight promote healing.

The term “clinical fracture-healing” refers to the absence of tendernesson manual palpation from fracture site. In some embodiments, the term“clinical fracture-healing” refers to no or mild pain (pain score 0-3)at the fracture site with full weight-bearing and pain will bedocumented using the visual analogue scale.

The term “radiographic fracture healing” refers to a condition that inview of the anteroposterior and lateral radiographs, investigatorsand/or an independent radiologist identified Cortical bridging and/ordisappearance of the fracture lines on 3 cortices of the 4 cortices atfracture site.

Assessment of Methods

Safety Assessment Methods

Adverse effect (AE): type, severity, management and outcome.

Systematic AE: any systematic sign, symptom, disease, laboratory testresult, radiographic finding, or physiologic observation that occurredor worsened after treatment, regardless of causality.

Local AE: including inflammation, infection (any suspected or confirmedsuperficial or deep infection involving soft tissue or bone, with orwithout bacteriologic confirmation), hardware failure, pain (new orincreased), peripheral edema, heterotopic ossification/soft-tissuecalcification, and complications related to wound-healing.

Efficacy Assessment Methods

The primary and secondary efficacy outcomes will be analyzed based onFull Analysis Set (FAS) and Per Protocol (PP) population. The primaryanalysis will be conducted on the FAS population.

The primary efficacy endpoint is the proportion of subjects who receivedsecondary intervention within 30 weeks after definitive wound closure.The primary analysis will be conducted on the FAS population usingCochran-Armitage trend test to indicate the linear trend in responserates with increasing homodimeric protein dosages. A supportive analysisusing the PP population will be performed for the primary efficacyendpoint.

In addition, the secondary efficacy endpoints will be analyzed orsummarized as below:

The proportion of subjects with clinical fracture healing and theproportion of subjects with radiographic healing within 30 weeks afterdefinitive wound closure will be compared separately usingCochran-Armitage trend test to indicate the linear trend in responserates with increasing homodimeric protein dosages.

The assessment of time from definitive wound closure to secondaryintervention, time from definitive wound closure to clinical fracturehealing and time from definitive wound closure to radiographic healingwill be summarized separately by group using descriptive statistics(Mean, SD).

The proportion of subjects who received secondary intervention, theproportion of subjects with clinical fracture healing and the proportionof subjects with radiographic healing within postoperative Week 6, Week12, Week 18, Week 24, Week 42 and Week 52 after definitive wound closurewill be summarized separately by group using descriptive statistics (n,%). If applicable, 95% CI of each group will be calculated based onClopper-Pearson exact CI method for a single binomial proportion.

Study Design 2

A Randomized, evaluator-blind, controlled study to evaluate the safetyand efficacy of three dosage levels of homodimeric protein (Hp)including the recombinant polypeptide produced according to Example 6(i.e., SEQ ID NO: 260)/β-TCP in combination with the cage and posteriorsupplemental fixation in patients with single level (between L1 to S1)degenerative disk disease (DDD) using posterior open approach for lumbarinterbody fusion. Subjects of 24 will be randomly assigned (1:1:1:1) to4 groups (1 control group and 3 different dose groups), and the clinicalstudy investigational device for treatment of each group are below:

Control group (6 subjects): standard of care (posterior open approachfor lumbar interbody fusion with cage) plus either autogenous bone graftimplantation (with or without β-TCP);

1 mg Hp/site (6 subjects): standard of care plus 1 mg homodimericprotein per site;

2 mg Hp/site (6 subjects): standard of care plus 2 mg homodimericprotein per site; and

3 mg Hp/site (6 subjects): standard of care plus 3 mg homodimericprotein per site.

Homodimeric protein will be supplied as 5.5 mg Hp/vial lyophilizedpowder with water for injections. After reconstitution, the homodimericprotein will be mixed with the β-TCP, with the final concentration of 1mg, 2 mg or 3 mg Hp/site. Then certain amount of the mixture will beapplied into the cage, which was determined by the size of cage used.

Homodimeric protein will be reconstituted (the exact volume of waterused to get the intended concentration will be stated in Table 18) inthree different concentration stock solutions and every 0.24 ml fromeach stock solution is required to mixed with block β-TCP (0.3 g) in thepolyetheretherketone (PEEK) cage (Wiltrom Co., Ltd./xxx series).

The final concentration of homodimeric protein applied at DDD site is:1.0; 2.0; 3.0 mg Hp/site.

Stock solution of homodimeric protein: 5.5 (mg)/1.32 (ml)=4.2 (mg/ml);

Final concentration of homodimeric protein (mg)/site: 4.2 (mg/ml)×0.24(ml)=1.0 mg

TABLE 18 Final Concentration Concen- Volume β-TCP Applied Hp/ Hp WFItration required required (Hp (mg)/ site (mg/vial) (mL) (mg/mL) (mL) (g)β-TCP (g)) (mg) 5.5 1.32 4.2 0.24 0.3 3.3 1.0 5.5 0.65 8.4 0.24 0.3 6.72.0 5.5 0.44 12.5 0.24 0.3 10.0 3.0

The source of autograft can be posterior superior iliac spine (PSIS) orbone chips obtained from posterior laminectomy. Autograft can be mixedwith β-TCP if the amount of autograft is insufficient. Unilateral orbilateral posterolateral fusion (can be one or two sides) with localgraft and posterior supplemental fixation measures can be used in allgroups based on investigator's judgment. Intravenous vancomycin (500 mgevery 6 hours) will be given prior to operation and for 3 consecutivedays.

Subjects will be followed for efficacy and safety for the main studyperiod of 24 weeks and an extension safety follow-up to 24 months afterindex surgery. In some embodiments, clinical investigators and anindependent evaluator will assess the efficacy by evaluating theradiographic results during the trial.

Inclusion Criteria:

Subjects will be included if ALL of the following inclusion criteriaapply:

The subject is ≥20 years old;

With single level DDD from L1 to S1 as noted by back pain of discogenicorigin, with or without radiculopathy secondary to nerve rootcompression, manifested by, history of radiating leg or buttock pain,paresthesias, numbness or weakness, or history of neurogenicclaudication;

Has radiographic evidence of advanced degenerative lumbosacral disease,such as decreased disk height; herniated nucleus pulposus; hypertrophyor thickening of the ligamentum flavum, annulus fibrosis, or facet jointcapsule; hypertrophied facet joints, facet joint space narrowing, orfacet periarticular osteophyte formation; trefoil canal shape; orlateral (subarticular) stenosis; or vertebral endplate osteophyteformation; and at least one of the following:

Sagittal plane translation (slippage) of the superior (cranial)vertebral body anterior or posterior to the inferior (caudal) vertebralbody is greater than 4 mm or angulation is greater than 10°, or Coronalplane translation (slippage) of the superior (cranial) vertebral bodylateral to the inferior (caudal) vertebral body is greater than 4 mm, ornarrowing (stenosis) of the lumbar spinal canal and/or intervertebralforamen;

Non responsive to non-operative treatment for at least 6 months;

Females of non-childbearing potential or who have a negative result onpregnancy test within 72 hours prior to surgery, or males;

Female subjects of childbearing potential (i.e., women who have not beensurgically sterilized or have not been post-menopausal for at least 1year) and male subject's partners of childbearing potential must agreeto use medically acceptable contraception methods throughout the studyperiod. Medically acceptable contraception methods include hormonalpatch, implant or injection intrauterine device, or double barriermethod (condom with foam or vaginal spermicidal suppository, diaphragmwith spermicidal). Complete abstinence can be considered an acceptablecontraception method. Oral contraceptive is an acceptable contraceptionmethod prior to the study, but an alternative method will be requiredduring the study;

If female, subject is not breast-feeding;

Willing to provide signed informed consent form (ICF) prior toparticipation in any study-related procedures and adhere to the studyrequirements for the length of the trial.

Exclusion Criteria:

Subjects will be excluded if ANY of the following exclusion criteriaapply:

Greater than Grade 1 spondylolisthesis (Meyerding's classification,refer to Appendix 1);

Spinal instrumentation implantation or interbody fusion procedurehistory at the involved level or vertebral body fracture at the plannedpedicle screw insertion level;

Established osteomalacia;

Active malignancy or prior malignancy history in past 5 years (exceptcured cutaneous basal cell carcinoma and cervical carcinoma in situ);

Active local or systemic infection;

Gross obesity, defined as BMI≥30;

Fever>38° C.;

Mentally incompetent. If questionable, obtain psychiatric consult;

Waddell Signs of Inorganic Behavior ≥3 (refer to Appendix 2);

Alcohol or drug abuse, as defined by currently undergoing treatment foralcohol and/or drug abuse. Alcohol abuse is a pattern of drinking thatresults in harm to one's health, interpersonal relationships, or abilityto work;

Autoimmune disease (e.g. Systemic Lupus Erythematosus ordermatomyositis);

Hypersensitivity to protein pharmaceuticals (monoclonal antibodies orgamma globulins);

Previous exposure to rhBMP-2;

Endocrine or metabolic disorders affecting osteogenesis (e.g., hypo- orhyper-thyroidism or parathyroidism, renal osteodystrophy, Ehlers-Danlossyndrome, or osteogenesis imperfecta);

Treatment for 7 days or more with prednisone [cumulative dose>150 mgwithin 6 months or other steroids with equivalent dose (refer toAppendix 3)], calcitonin (within 6 months). Treatment of Bisphosphonates(for 30 days or more within 12 months), therapeutic doses of fluoride(for 30 days within 12 months), and anti-neoplastic, immunostimulatingor immunosuppressive agents within 30 days prior to implantation of theassigned treatment;

Treatment with any investigational therapy within 28 days ofimplantation surgery;

Has scoliosis greater than 30 degrees;

Subjects have the history or clinical manifestations of significant CNS,cardiovascular, pulmonary, hepatic, renal, metabolic, gastrointestinal,urological, endocrine or hematological disease;

Has a medical disease or condition that would preclude accurate clinicalevaluation of the safety and effectiveness of the treatments in thisstudy, such as motor weakness, sensory loss, or painful conditions thatinhibit normal ambulation or other activities of daily living;

Has abnormal renal and/or hepatic functions, with Creatinine or ALT orAST value>5 times the upper normal limit;

Has a documented allergy or intolerance to PEEK;

History of hypersensitivity or allergy to Vancomycin;

Any condition that it is not suitable for subjects to participate in thestudy based on the physician's judgement.

Planned Study Duration:

Screening period: 14 days. Ensure the subject has signed the ICF andassess whether the subject is eligible for the study. The assessmentsinclude physical examination, vital signs, electrocardiogram, blood orurine pregnancy test, laboratory examination, pre-operative clinical andradiology evaluation. The data of demography, medical history,concomitant medication and adverse events should be collected.

Treatment period: 1 day. Check whether the subject is eligible for thestudy, obtain the baseline sample/data and administrate theinvestigational products. The assessments include physical examination,vital signs and radiology examination. The data of operationinformation, concomitant medication and adverse events should becollected.

Follow-up period: Subjects will be followed for the main study period of24 weeks and an extension safety follow-up to 24 months after theimplantation. Perform the assessments at Week 6, Week 12, Week 18, Week24, Month 12, Month 18, Month 24 after treatment. The assessmentsinclude concomitant treatments, physical examination, laboratoryevaluations, vital signs and radiographic examination(anterior/posterior and lateral views, flexion/extension films). Highresolution thin-slice CT scans (1 mm slices with 1 mm index on axialsagittal and coronal reconstructions) will be performed at Week 24 andMonth 24.

Assessments of Efficacy

Primary Endpoint:

The primary study efficacy endpoint is the proportion of subjects havingfusion success at postoperative Week 24.

Secondary Endpoints:

The proportion of subjects having fusion success at postoperative Month12, Month 18, and Month 24.

Time from baseline to radiographic fusion.

The proportion of subjects who have additional surgicalprocedures/interventions within postoperative Week 24, Month 12, Month18 and Month 24; the operation time (from skin incision to woundclosure), blood loss (during the operation) and hospital stay will berecorded.

Success rate of Oswestry Disability Index (ODI, refer to Appendix 4) atpostoperative Week 24, Month 12, Month 18 and Month 24; ODIQuestionnaire was used to assess patient back function. The ODI scoreranges from 0-100. The best score is 0 (no disability) and the worst is100 (maximum disability). Success rate of ODI is reported as percent ofsubjects whose ODI score met: pre-operative score−post-operativescore≥15.

Success rate of improvement in Visual Analogous Scale (VAS, refer toAppendix 5) at postoperative Week 24, Month 12, Month 18 and Month 24.Success rate of back pain is reported as percent of subjects whoseimprovement in back pain met: pre-operative score−post-operativescore>0. Success rate of leg pain is reported as percent of subjectswhose improvement in leg pain met: pre-operative score−post-operativescore>0.

Efficacy Analyses

The primary and secondary efficacy outcomes will be analyzed based onFull Analysis Set (FAS) and Per Protocol (PP) population. The primaryanalysis will be conducted on the FAS population.

The primary study efficacy endpoint is the proportion of subjects havingfusion success at postoperative Week 24. The primary analysis will beconducted on the FAS population by using Cochran-Armitage trend test toindicate the linear trend in response rates with increasing homodimericprotein dosages. A supportive analysis for the PP population will beperformed for the primary efficacy endpoint.

In addition, the secondary efficacy endpoints will be analyzed orsummarized as below:

The proportion of subjects having fusion success at postoperative Month12, Month 18, Month 24 and the proportion of subjects who haveadditional surgical procedures/interventions within postoperative Week24, Month 12, Month 18 and Month 24 will be compared separately by usingCochran-Armitage trend test to indicate the linear trend in responserates with increasing homodimeric protein dosages.

The assessment of time from baseline to radiographic fusion will besummarized by group using descriptive statistics (Mean, SD).

The assessment of operation time (from skin incision to wound closure),blood loss (during the operation) and hospital stay will be summarizedseparately by arm using descriptive statistics (Mean, SD).

Success rate of ODI at postoperative Week 24, Month 12, Month 18 andMonth 24 and success rate of VAS at postoperative Week 24, Month 12,Month 18 and Month 24 will be summarized separately by arm usingdescriptive statistics (n, %). If applicable, 95% CI of each arm will becalculated based on Clopper-Pearson exact CI method for a singlebinomial proportion.

The disclosure is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the disclosure inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

Other embodiments are within the following claims.

What is claimed is:
 1. A biodegradable composition, comprising: arecombinant polypeptide comprising: a first domain selected from thegroup consisting of SEQ ID NO: 35 and SEQ ID NO: 39; a second domainselected from the group consisting of SEQ ID NO: 47 and SEQ ID NO: 49;and a third domain selected from the group consisting of SEQ ID NO: 57and SEQ ID NO: 61; wherein the first domain is fused to eitherC-terminal or N-terminal of the second domain, the third domain is fusedto the second domain or the first domain, and wherein the second domaincomprises an intramolecular disulfide bond and the recombinantpolypeptide is capable of inducing alkaline phosphatase activity; and abiodegradable calcium phosphate carrier having a plurality of pores. 2.The biodegradable composition of claim 1, wherein the third domaincomprises a first amino acid sequence of PKACCVPTE (SEQ ID NO: 356) anda second amino acid sequence of GCGCR (SEQ ID NO: 357), and wherein thethird domain comprises two intramolecular disulfide bonds between thefirst and second amino acid sequences.
 3. The biodegradable compositionof claim 2, wherein the recombinant polypeptide comprises: (a) a firstintramolecular disulfide bond between the fourth amino acid of the firstamino acid sequence and the second amino acid of the second amino acidsequence, and a second intramolecular disulfide bond between the fifthamino acid of the first amino acid sequence and the fourth amino acid ofthe second amino acid sequence, or (b) a first intramolecular disulfidebond between the fifth amino acid of the first amino acid sequence andthe second amino acid of the second amino acid sequence, and a secondintramolecular disulfide bond between the fourth amino acid of the firstamino acid sequence and the fourth amino acid of the second amino acidsequence.
 4. A biodegradable composition, comprising: a recombinantpolypeptide comprising an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 260, SEQ ID NO: 292, SEQ ID NO: 324, and SEQID NO: 332, wherein the recombinant polypeptide comprises anintramolecular disulfide bond between cysteine 44 and cysteine 48 and iscapable of inducing alkaline phosphatase activity; and a biodegradablecalcium phosphate carrier having a plurality of pores.
 5. Abiodegradable composition capable of inducing bone growth to form a bonemass in a location, comprising: a homodimeric protein, comprising: twoidentical recombinant polypeptides each comprising: a first domainselected from the group consisting of SEQ ID NO: 35 and SEQ ID NO: 39; asecond domain selected from the group consisting of SEQ ID NO: 47 andSEQ ID NO: 49; and a third domain selected from the group consisting ofSEQ ID NO: 57 and SEQ ID NO: 61; wherein the first domain is fused toeither C-terminal or N-terminal of the second domain, the third domainis fused to the second domain or the first domain, and wherein thesecond domain comprises an intramolecular disulfide bond, wherein thehomodimeric protein comprises an intermolecular disulfide bond betweenthe first domains of the two recombinant polypeptides; and abiodegradable calcium phosphate carrier having a plurality of pores. 6.The biodegradable composition of claim 5, wherein the third domain ofeach recombinant polypeptide comprises a first amino acid sequence ofPKACCVPTE (SEQ ID NO: 356) and a second amino acid sequence of GCGCR(SEQ ID NO: 357), and wherein the homodimeric protein comprises twointermolecular disulfide bonds between the first amino acid sequence inthe third domain of one recombinant polypeptide and the second aminoacid sequence in the third domain of the other recombinant polypeptide.7. The biodegradable composition of claim 6, wherein the homodimericprotein comprises: (a) a first intermolecular disulfide bond between thefourth amino acid of the first amino acid sequence of the onerecombinant polypeptide and the second amino acid of the second aminoacid sequence of the other recombinant polypeptide, and a secondintermolecular disulfide bond between the fifth amino acid of the firstamino acid sequence of the one recombinant polypeptide and the fourthamino acid of the second amino acid sequence of the other recombinantpolypeptide, or (b) a first intermolecular disulfide bond between thefifth amino acid of the first amino acid sequence of the one recombinantpolypeptide and the second amino acid of the second amino acid sequenceof the other recombinant polypeptide, and a second intermoleculardisulfide bond between the fourth amino acid of the first amino acidsequence of the one recombinant polypeptide and the fourth amino acid ofthe second amino acid sequence of the other recombinant polypeptide. 8.The biodegradable composition of claim 5, wherein the homodimericprotein is about 0.003-0.32% (w/w).
 9. The biodegradable composition ofclaim 8, wherein a porosity of the biodegradable calcium phosphatecarrier is larger than 70% with pore size from about 300 pm to about 600μm.
 10. The biodegradable composition of claim 8, wherein the pluralityof pores extend throughout the biodegradable calcium phosphate carrier,and wherein the homodimeric protein is in an effective amount of fromabout 0.03 mg/g to about 3.2 mg/g of the biodegradable calcium phosphatecarrier.
 11. The biodegradable composition of claim 8, wherein thebiodegradable composition is suitable for augmentation of a tissueselected from nasal furrows, frown lines, midfacial tissue, jaw-line,chin, and cheeks, and wherein the location is selected from the groupsconsisting of a long-bone fracture defect, a space between two adjacentvertebra bodies, a non-union bone defect, maxilla osteotomy incision,mandible osteotomy incision, sagittal split osteotomy incision,genioplasty osteotomy incision, rapid palatal expansion osteotomyincision, and a space extending lengthwise between two adjacenttransverse processes of two adjacent vertebrae.
 12. The biodegradablecomposition of claim 8, wherein a single dose of the homodimeric proteinis from about 0.006 mg to about 15 mg.
 13. A method of promoting healingof a long-bone fracture in a subject in need, comprising: preparing thebiodegradable composition of claim 5, wherein the homodimeric protein ishomogeneously entrained within the biodegradable calcium phosphatecarrier that hardens so as to be impermeable to efflux of thehomodimeric protein in vivo sufficiently that the long-bone fracturehealing is confined to the volume of the biodegradable calcium phosphatecarrier; and implanting the biodegradable composition at a locationwhere the long-bone fracture occurs, wherein the homodimeric protein isin an amount of from about 0.03 mg/g to about 3.2 mg/g of thebiodegradable calcium phosphate carrier.
 14. A method of promotingspinal fusion in a subject in need, comprising: exposing an uppervertebra and a lower vertebra of the subject, identifying a site forfusion between the upper and the lower vertebra, exposing a bone surfaceon each of the upper and the lower vertebra at the site for fusion, andadministering the biodegradable composition of claim 5 to the site forfusion.
 15. The method of claim 14, wherein the biodegradable calciumphosphate carrier is a non-compressible delivery vehicle, and thenon-compressible delivery vehicle is for application to the site forfusion between the two bone surfaces where bone growth is desired butdoes not naturally occur, and the site for fusion extends lengthwisebetween two adjacent transverse processes of the upper and the lowervertebrae.
 16. A spinal fusion device, comprising: the biodegradablecomposition of claim 5; and a spinal fusion cage, configured to retainthe biodegradable calcium phosphate carrier.
 17. A method of generatinga bone mass to fuse two adjacent vertebrae bodies in a spine of asubject in need, comprising introducing the biodegradable composition ofclaim 5 in a location between the two adjacent vertebrae bodies, whereinthe homodimeric protein is in an amount of from about 0.2 mg/site toabout 10.5 mg/site of the location.
 18. A moldable composition forfilling an osseous void, comprising: about 90% to about 99.5% by weightof a moldable matrix; and a homodimeric protein comprising: twoidentical recombinant polypeptides each comprising: a first domainselected from the group consisting of SEQ ID NO: 35 and SEQ ID NO: 39; asecond domain selected from the group consisting of SEQ ID NO: 47 andSEQ ID NO: 49; and a third domain selected from the group consisting ofSEQ ID NO: 57 and SEQ ID NO: 61; wherein the first domain is fused toeither C-terminal or N-terminal of the second domain, the third domainis fused to the second domain or the first domain, and wherein thesecond domain comprises an intramolecular disulfide bond, wherein thehomodimeric protein comprises an intermolecular disulfide bond betweenthe first domains of the two recombinant polypeptides, wherein less thanabout 25% by percentage of the homodimeric protein is released from themoldable composition after a predetermined hour post implantation.